Drug delivery product and methods

ABSTRACT

The present invention provides a particulate delivery system comprising an extracted yeast cell wall comprising beta-glucan, a payload molecule and a payload trapping molecule. The invention further provides methods of making and methods of using the particulate delivery system.

BACKGROUND OF THE INVENTION

Drug delivery systems are designed to provide a biocompatible reservoirof an active agent for the controlled release of the active agentdependent either on time, or on local conditions, such as pH. Whilemacroscopic drug delivery systems such as transdermal patches,implantable osmotic pumps and implantable subcutaneous depots (e.g.,NORPLANT™) have had some success, there has been continuing interest inmicroscopic drug delivery systems such as microcapsules, microparticlesand liposomes.

Microcapsules and microspheres are usually powders consisting ofspherical particles 2 millimeters or less in diameter, usually 500microns or less in diameter. If the particles are less than 1 micron,they are often referred to as nanocapsules or nanospheres. A descriptionof methods of making and using microspheres and microcapsules can befound, for example in U.S. Pat. No. 5,407,609. Microcapsules andmicrospheres can be distinguished from each other by whether the activeagent is formed into a central core surrounded by an encapsulatingstructure, such as a polymeric membrane, or whether the active agent isdispersed throughout the particle; that is, the internal structure is amatrix of the agent and excipient, usually a polymeric excipient. Therelease of the active agent from a microcapsule is often regulated bythe biodegradation of the matrix material, usually a biodegradablepolymeric material such as either poly(DL-lactide) (DL-PL) orpoly(DL-lactide-co-glycolide) (DL-PLG) as the polymeric excipient.

Liposomes can be considered microcapsules in which the active agent coreis encompassed by a lipid membrane instead of a polymeric membrane.Liposomes are artificial lipid vesicles consisting of lipid layers,where the antigen may be encapsulated inside the aqueous compartment ofthe liposome, or associated with the antigen on the surface viasurface-coupling techniques. Liposomes can be prepared easily andinexpensively on a large scale and under conditions that are mild toentrapped antigens. They do not induce immune responses to themselves,and are used in humans for parenterally administered drugs.

While the high surface area/volume ratio of microcapsules, microspheresand liposomes favor the release of the active agent, their small sizeprovides challenges in manufacturing. A wide variety of methods toprepare microcapsules and microspheres are described in the literature,e.g., U.S. Pat. No. 5,407,609. Several of these methods make use ofemulsions to make microspheres, in particular to make microspheres lessthan 2 millimeters in diameter. To give a general example of suchprocesses, one can dissolve a polymer in a suitable organic solvent (thepolymer solvent), dissolve or disperse an agent in this polymersolution, disperse the resulting polymer/agent mixture into an aqueousphase (the processing medium) to obtain an oil-in-water emulsion withoil microdroplets dispersed in the processing medium, and remove thesolvent from the microdroplets to form microspheres. These processes canalso be performed with water-in-oil emulsions and with double emulsions.The use of emulsion-based processes that follow this basic approach isdescribed in several U.S. patents, such as U.S. Pat. Nos. 3,737,337,3,891,570, 4,384,975, 4,389,330, and 4,652,441.

Alternatively, extracted yeast cell wall particles are readilyavailable, biodegradable, substantially spherical particles about 2-4 μmin diameter. Preparation of extracted yeast cell wall particles is knownin the art, and is described, for example in U.S. Pat. Nos. 4,992,540,5,082,936, 5,028,703, 5,032,401, 5,322,841, 5,401,727, 5,504,079,5,968,811, 6,444,448 B1, 6,476,003 B 1, published U.S. applications2003/0216346 A1, 2004/0014715 A1, and PCT published application WO02/12348 A2. A form of extracted yeast cell wall particles, referred toas “whole glucan particles,” have been suggested as delivery vehicles,but have been limited either to release by simple diffusion of activeingredient from the particle or release of an agent chemicallycrosslinked to the whole glucan particle by biodegradation of theparticle matrix. See U.S. Pat. Nos. 5,032,401 and 5,607,677.

Extracted yeast cell wall particles, primarily due to their beta-glucancontent, are targeted to phagocytic cells, such as macrophages and cellsof lymphoid tissue. The mucosal-associated lymphoid tissue (MALT)comprises all lymphoid cells in epithelia and in the lamina proprialying below the body's mucosal surfaces. The main sites ofmucosal-associated lymphoid tissues are the gut-associated lymphoidtissues (GALT), and the bronchial-associated lymphoid tissues (BALT).

Another important component of the GI immune system is the M ormicrofold cell. M cells are a specific cell type in the intestinalepithelium over lymphoid follicles that endocytose a variety of proteinand peptide antigens. Instead of digesting these proteins, M cellstransport them into the underlying tissue, where they are taken up bylocal dendritic cells and macrophages.

M cells take up molecules and particles from the gut lumen byendocytosis or phagocytosis. This material is then transported throughthe interior of the cell in vesicles to the basal cell membrane, whereit is released into the extracellular space. This process is known astranscytosis. At their basal surface, the cell membrane of M cells isextensively folded around underlying lymphocytes and antigen-presentingcells, which take up the transported material released from the M cellsand process it for antigen presentation.

A study has shown that transcytosis of yeast particles (3.4+/−0.8 micronin diameter) by M cells of the Peyer's patches takes less than 1 hour(Beier, R., & Gebert, A., Kinetics of particle uptake in the domes ofPeyer's patches, Am J. Physiol. 1998 July; 275(1 Pt L):G130-7). Withoutsignificant phagocytosis by intraepithelial macrophages, the yeastparticles migrate down to and across the basal lamina within 2.5-4hours, where they quickly get phagocytosed and transported out of thePeyer's patch domes. M cells found in human nasopharyngeal lymphoidtissue (tonsils and adenoids) have been shown to be involved in thesampling of viruses that cause respiratory infections. Studies of an invitro M cells model have shown uptake of fluorescently labeledmicrospheres (Fluospheres, 0.2 μm) and chitosan microparticles (0.2 μm)van der Lubben I. M., et al., Transport of chitosan microparticles formucosal vaccine delivery in a human intestinal M-cell model, J DrugTarget, 2002 September; 10(6):449-56. A lectin, Ulex europaeusagglutinin 1 (UEA1, specific for alpha-L-fucose residues) has been usedto target either polystyrene microspheres (0.5 nm) or polymerizedliposomes to M cells (0.2 μm) (Clark, M. A., et al., Targetingpolymerised liposome vaccine carriers to intestinal M cells, Vaccine.2001 Oct. 12; 20(1-2):208-17). In vivo studies in mice have reportedthat poly-D,L-lactic acid (PDLLA) microspheres or gelatin microspheres(GM) can be efficiently taken up by macrophages and M cells. (Nakase,H., et al., Biodegradable microspheres targeting mucosalimmune-regulating cells: new approach for treatment of inflammatorybowel disease, J. Gastroenterol. 2003 March, 38 Suppl 15:59-62).

However, it has been reported that uptake of synthetic particulatedelivery vehicles including poly(DL-lactide-co-glycolide) microparticlesand liposomes is highly variable, and is determined by the physicalproperties of both particles and M cells. Clark, M. A., et al.,Exploiting M cells for drug and vaccine delivery, Adv Drug Deliv Rev.2001 Aug. 23; 50(1-2):81-106. The same study reported that delivery maybe enhanced by coating the particles or liposomes with reagentsincluding appropriate lectins, microbial adhesins and immunoglobulinswhich selectively bind to M cell surfaces. See also, Florence, A. T.,The oral absorption of micro- and nanoparticulates: neither exceptionalnor unusual, Pharm Res. 1997 March; 14(3):259-66.

Pathogen pattern recognition receptors (PRRs) recognize commonstructural and molecular motifs present on microbial surfaces andcontribute to induction of innate immune responses. Mannose receptorsand beta-glucan receptors in part participate in the recognition offungal pathogens. The mannose receptor (MR), a carbohydrate-bindingreceptor expressed on subsets of macrophages, is considered one suchPRR. Macrophages have receptors for both mannose and mannose-6-phosphatethat can bind to and internalize molecules displaying these sugars. Themolecules are internalized by endocytosis into a pre-lysosomal endosome.This internalization has been used to enhance entry of oligonucleotidesinto macrophages using bovine serum albumin modified withmannose-6-phosphate and linked to an oligodeoxynucleotide by a disulfidebridge to a modified 3′ end; see Bonfils, E., et al., Nucl. Acids Res.1992 20, 4621-4629. see E. Bonfils, C. Mendes, A. C. Roche, M. Monsignyand P. Midoux, Bioconj. Chem., 3, 277-284 (1992). Macrophages alsoexpress beta-glucan receptors, including CR3 (Ross, G. D., J. A. Cain,B. L. Myones, S. L. Newman, and P. J. Lachmann. 1987. Specificity ofmembrane complement receptor type three (CR₃) for β-glucans. ComplementInflamm. 4:61), dectin-1. (Brown, G. D. and S. Gordon. 2001. Immunerecognition. A new receptor for J-glucans. Nature 413:36), andlactosylceramide (Zimmerman J W, Lindermuth J, Fish P A, Palace G P,Stevenson T T, DeMong D E. A novel carbohydrate-glycosphinglipidinteraction between a beta-(1-3)-glucan immunomodulator, PGG-glucan, andlactosylceramide of human leukocytes. J Biol. Chem. 1998 Aug.21:273(34):22014-20). The beta-glucan receptor, CR₃ is predominantlyexpressed on monocytes, neutrophils and NK cells, whereas dectin-1 ispredominantly expressed on the surface of cells of the macrophages.Lactosylceramide is found at high levels in M cells. Microglia can alsoexpress a beta-glucan receptor (Muller, C. D., et al. Functionalbeta-glucan receptor expression by a microglial cell line, Res Immunol.1994 May; 145(4):267-75).

There is evidence for additive effects on phagocytosis of binding toboth mannose and beta-glucan receptors. Giaimis et al. reportedobservations suggesting that phagocytosis of unopsonized heat-killedyeast (S. cerevisiae) by murine macrophage-like cell lines as well asmurine peritoneal resident macrophages is mediated by both mannose andbeta-glucan receptors. To achieve maximal phagocytosis of unopsonizedheat-killed yeast, coexpression of both mannose and beta-glucanreceptors is required (Giaimis, J., et al., Both mannose and beta-glucanreceptors are involved in phagocytosis of unopsonized, heat-killedSaccharomyces cerevisiae by murine macrophages, J Leukoc Biol. 1993December; 54(6):564-71).

SUMMARY OF THE INVENTION

In a preferred embodiment, the present invention provides a particulatedelivery system comprising an extracted yeast cell wall comprisingbeta-glucan and a payload trapping molecule. The particulate deliverysystem optionally, but typically, also includes a payload molecule,wherein the payload molecule and the payload trapping molecule aresoluble in the same solvent system. In preferred embodiments, thesolvent system comprises water. In other preferred embodiments, thesolvent system consists essentially of water. The particulate deliverysystem of the present invention is useful for both in vivo and in vitrodelivery of payload molecules to cells.

In particularly preferred embodiments, extracted yeast cell wallcomprises less than 90 weight percent beta-glucan. In certain preferredembodiments, the extracted yeast cell wall comprises more than 50 weightpercent chitin. In other preferred embodiments, the extracted yeast cellwall further comprises more than 30 weight percent mannan. In certainembodiments, the extracted yeast cell wall includes more than 1 weightpercent protein.

In preferred embodiments, the payload molecule is selected from thegroup consisting of a polynucleotide, a peptide, a protein, a smallorganic active agent, a small inorganic active agent and a mixturethereof. In certain preferred embodiments, the payload molecule is apolynucleotide selected from the group consisting of an oligonucleotide,an antisense construct, a siRNA, an enzymatic RNA, a recombinant DNAconstruct, an expression vector, and a mixture thereof. In otherpreferred embodiments, the particulate delivery system of the presentinvention is useful for in vivo or in vitro delivery of payloadmolecules such as amino acids, peptides and proteins. The peptides canbe signaling molecules such as hormones, neurotransmitters orneuromodulators, and can be the active fragments of larger molecules,such as receptors, enzymes or nucleic acid binding proteins. Theproteins can be enzymes, structural proteins, signaling proteins ornucleic acid binding proteins, such as transcription factors.

In other preferred embodiments, the payload molecule is a small organicactive agent, such as a therapeutic agent or a diagnostic agent. Inparticularly preferred embodiments, the small organic active agent is asequence-specific DNA binding oligomer, more preferably an oligomer ofheterocyclic polyamides that bind to the minor groove of double strandedDNA, such as those disclosed in U.S. Pat. No. 6,506,906 and in Dervan,P. Molecular Recognition of DNA by Small Molecules, Bioorganic &Medicinal Chemistry (2001) 9: 2215-2235, both of which are herebyincorporated by reference. In preferred embodiments, the oligomer hasmonomeric subunits selected from the group consisting ofN-methylimidazole carboxamide, N-methylpyrrole carboxamide, beta-alanineand dimethylaminopripylamide.

In other preferred embodiments, the particulate delivery system of thepresent invention includes inorganic active agents, e.g.,gastrointestinal therapeutic agents such as aluminum hydroxide, calciumcarbonate, magnesium carbonate, sodium carbonate and the like.

The choice of the payload trapping molecule can confer specificcharacteristics to the particulate delivery system. In general, thepreferred payload trapping molecule is biocompatible andpharmaceutically acceptable. As noted above, the payload molecule andthe payload trapping molecule are soluble in the same solvent system.Suitable payload trapping molecules include natural and syntheticpolymers. In certain embodiments, the physical characteristics of thepayload trapping molecule, such as agarose or polyacrylamide, provideuseful advantages

Suitable polymers include polysaccharides. In preferred embodiments, thepolysaccharide selected is from the group consisting of agarose, analginate, a xanthan, a dextran, a chitosan, a galactomannan gum, aderivative thereof and a mixture thereof. In certain preferredembodiments, the polysaccharides have been derivatized to producecationic or anionic characteristics at physiological pH.

In other embodiments, the payload trapping molecule is a chargedmolecule at physiological pH, such as a cationic polymer, an anionicpolymer, a cationic detergent, an anionic detergent and a mixturethereof. Preferred cationic polymers include chitosan, poly-L-lysine andpolyethylenimines (PEIs), including substantially linearpolyethylenimines, such as JetPEI, a commercially available linearpolyethylenimine cationic polymer transfection reagent (Qbiogene, Inc.,CA). Other cationic polymer transfection reagents are also suitable,preferably CytoPure™, a proprietary, commercially available,water-soluble cationic polymer transfection reagent (Qbiogene, Inc.,CA). In other preferred embodiments, suitable anionic polymers includealginates, dextrans and xanthans, including derivatized alginates,dextrans and xanthans. In further preferred embodiments, the payloadtrapping molecule is a cationic detergent such ashexadecyltrimethylammoniumbromide. In one preferred embodiment, amixture of a cationic detergent, such ashexadecyltrimethylammoniumbromide, and a cationic polymer, such as apolyethylenimine, is used. In another preferred embodiment, a mixture ofa cationic detergent, such as hexadecyltrimethylammoniumbromide, and acationic polymer, such as chitosan or PEI, can be used.

While not being held to a single hypothesis, it is believed that, inaddition to facilitating the retention of the payload by the yeast cellwall particles, a preferred payload trapping molecule serves toencourage the release of the payload molecule from the endosome of aphagocytic cell by acting as a detergent, by helping to swell theendosome osmotically, or by other effects.

In other preferred embodiments, the present invention provides methodsof using the particulate delivery system. In certain preferredembodiments, the invention provides methods of delivering a payloadmolecule to a cell comprising the steps of providing a extracted yeastcell wall comprising beta-glucan, the yeast cell wall defining aninternal space; contacting the extracted yeast cell wall with a payloadmolecule wherein the payload molecule becomes at least partiallyenclosed within the internal space; contacting the extracted yeast cellwall with a payload trapping molecule wherein the payload trappingmolecule at least partially confines the payload molecule within theextracted yeast cell wall to form a particulate delivery system; andcontacting a cell with the particulate delivery system. Preferably themethod further includes the step of internalizing the particulatedelivery system by the cell.

In other preferred embodiments, the invention provides methods of makinga particulate delivery system comprising the steps of providing aextracted yeast cell wall comprising beta-glucan, the yeast cell walldefining an internal space; contacting the extracted yeast cell wallwith a payload molecule wherein the payload molecule becomes associatedwith the extracted yeast cell wall; contacting the extracted yeast cellwall with a payload trapping molecule wherein the payload trappingmolecule stabilizes the association of the payload molecule with theextracted yeast cell wall to form a particulate delivery system. Inpreferred embodiments, the method also includes the steps of washing anddrying the particulate delivery system.

In other preferred embodiments, the present invention provides methodsof exposing an individual to an antigen comprising the step ofcontacting a phagocytic cell of the individual with a particulatedelivery system comprising an extracted yeast cell wall comprisingbeta-glucan, a payload trapping molecule and payload molecule, whereinthe payload molecule is a polynucleotide comprising a control elementoperatively linked to an open reading frame encoding a peptide that canbe controllably expressed in the cells of the individual. Preferably theencoded peptide is an antigenic peptide. In further preferredembodiments, the present invention provides methods of exposing anindividual to an antigen comprising the step of contacting a phagocyticcell of the individual with a particulate delivery system comprising anextracted yeast cell wall comprising beta-glucan, a payload trappingmolecule and payload molecule, wherein the payload molecule is aantigenic molecule. A preferred antigenic molecule is a toxoid.

In preferred embodiments, the contacted cells are macrophages, but mayalso include any cell capable of yeast particle phagocytosis, includingM cells of the Peyer's patches, monocytes, neutrophils, dendritic cells,Langerhans cells, Kupffer cells, alveolar phagocytes, peritonealmacrophages, milk macrophages, microglia, eosinophils, granulocytes,mesengial phagocytes, synovial A cells and other phagocytes. Inpreferred embodiments, the particulate delivery system is administeredorally and absorbed via M cells of the Peyer's patches in the gut.

In preferred embodiments the polynucleotide is a recombinant DNAconstruct comprising a control element operatively linked to an openreading frame encoding a protein, e.g. an expression vector. The proteincan be a structural protein, a protein having enzymatic activity, amembrane protein a DNA binding protein or a signaling protein. Incertain preferred embodiments, the protein is an antigenic protein.

In certain preferred embodiments, the method further includes the stepof the cell expressing the protein. The expressed protein can beretained intracellularly by the cell, incorporated in a membranousstructure, such as the plasma membrane, or be secreted by the cell.

In other embodiments, more than one type of polynucleotide is enclosedwithin the particulate delivery system. In preferred embodiments, thepolynucleotides provide the ability to express multiple gene productsunder control. In certain embodiments, at least one expressible geneproduct is a membrane protein, preferably a membrane receptor, mostpreferably a membrane-bound receptor for a signaling molecule. In someembodiments, at least one expressible gene product is a soluble protein,preferably a secreted protein, most preferably a signaling protein orpeptide.

In other embodiments, the present invention provides a method ofdelivering a drug to a macrophage cell including the steps of providinga substantially spherical extracted yeast cell wall comprisingbeta-glucan, the yeast cell wall defining an internal space; contactingthe extracted yeast cell wall with a drug wherein the drug is at leastpartially enclosed within the internal space; contacting the extractedyeast cell wall with a trapping molecule wherein the trapping moleculeis at least partially enclosed within the internal space to form aparticulate drug delivery system; and contacting a macrophage cell withthe particulate drug delivery system. Preferably, the method alsoincludes the step of internalizing the particulate drug delivery systemby the macrophage. In preferred embodiments, the method also includesthe step of transporting the particulate drug delivery system by themacrophage. In particularly preferred embodiments, the macrophagedelivers the particulate drug delivery system to a macrophage-attractingsite, such as a site of infection, inflammatory reaction, hypoxia orhyperplasia. In certain preferred embodiments, the macrophage deliversthe particulate drug delivery system to a tumor. In particularlypreferred embodiments, the method includes the step of releasing thedrug from the particulate drug delivery system, more preferably furtherincluding the step of releasing the drug into the extracellular space.In certain embodiments, the step of releasing the drug includes thesteps of expressing a recombinant protein and secreting the protein intothe extracellular space.

The present invention provides a method of immunizing an individualagainst a pathogen. The method comprises the step of contacting cells ofsaid individual with a particulate delivery system comprising anextracted yeast cell wall comprising beta, glucan, a payload trappingmolecule and a nucleic acid composition, thereby administering to thecells a nucleic acid molecule that comprises a nucleotide sequence thatencodes a peptide which comprises at least an epitope identical to, orsubstantially similar to an epitope displayed on said pathogen asantigen, and said nucleotide sequence is operatively linked toregulatory sequences, wherein the nucleic acid molecule is capable ofbeing expressed in the cells of the individual.

In another preferred embodiment, the present invention provides a methodof producing immunity to a toxoid comprising the steps of providing aparticulate delivery system comprising an extracted yeast cell wallcomprising beta-glucan, a payload trapping molecule and a toxoid,contacting a phagocytic cell with the particulate delivery system andinducing phagocytosis of the particulate delivery system. The phagocyticcell can be one or more of macrophages, M cells of the Peyer's patches,monocytes, neutrophils, dendritic cells, Langerhans cells, Kupffercells, alveolar phagocytes, peritoneal macrophages, milk macrophages,microglia, eosinophils, granulocytes, mesengial phagocytes, and synovialA cells.

The present invention provides methods of immunizing an individualagainst a hyperproliferative disease or an autoimmune disease. Themethods comprise the step of contacting cells of said individual with aparticulate delivery system comprising an extracted yeast cell wallcomprising beta-glucan, a payload trapping molecule which includes anucleic acid composition, thereby administering to the cells a nucleicacid molecule that comprises a nucleotide sequence that encodes apeptide which comprises at least an epitope identical to, orsubstantially similar to an epitope displayed on a hyperproliferativedisease-associated protein or an autoimmune disease-associated protein,respectively, and is operatively linked to regulatory sequences, whereinthe nucleic acid molecule is capable of being expressed in the cells ofthe individual.

The present invention also provides methods of treating an individualsuffering from an autoimmune disease comprising the steps of contactingcells said individual with a particulate delivery system comprising anextracted yeast cell wall comprising beta-glucan, a payload trappingmolecule which includes a nucleic acid composition, therebyadministering to the cells a nucleic acid molecule that comprises anucleotide sequence that restores the activity of an absent, defectiveor inhibited gene, or that encodes a protein that produces a therapeuticeffect in the individual, and is operatively linked to regulatorysequences; the nucleic acid molecule being capable of being expressed insaid cells.

In further embodiments, the present invention provides a method ofimmunizing an individual against a hyperproliferative disease comprisingthe step of contacting cells of said individual with a particulatedelivery system comprising an extracted yeast cell wall comprisingbeta-glucan, a payload trapping molecule and a payload molecule that isa polynucleotide comprising a control sequence operatively linked to anopen reading frame encoding a peptide that comprises an epitopeidentical to, or substantially similar to, an epitope displayed on ahyperproliferative disease-associated protein, wherein encoded peptideis capable of being expressed in the cells of the individual. In otherembodiments, the present invention provides a method of treating anindividual suffering from a genetic disease comprising the step ofcontacting cells of said individual with a particulate delivery systemcomprising an extracted yeast cell wall comprising beta-glucan, apayload trapping molecule and a payload molecule that is apolynucleotide thereby administering to the cells a polynucleotide thatcomprises a nucleotide sequence that restores the activity of an absent,defective or inhibited gene. Preferably, the polynucleotide comprises aregulatory sequence operatively linked to an open reading frame encodinga protein that produces a therapeutic effect in the individual, theprotein being capable of being expressed in said cells.

The present invention also relates to methods of treating an individualsuffering from an autoimmune disease comprising the steps of contactingcells said individual with a particulate delivery system comprising anextracted yeast cell wall comprising beta-glucan, a payload trappingmolecule which includes a nucleic acid composition, therebyadministering to the cells a nucleic acid molecule that comprises anucleotide sequence that restores the function of an absent, defectiveor inhibited gene, or that encodes a protein that produces a therapeuticeffect in the individual, and is operatively linked to regulatorysequences; the nucleic acid molecule being capable of being expressed insaid cells.

Accordingly the present invention provides compositions and methodswhich prophylactically and/or therapeutically immunize an individualagainst a pathogen or abnormal, disease-related cell. The geneticmaterial encodes a peptide or protein that shares at least an epitopewith an immunogenic protein found on the pathogen or cells to betargeted. The genetic material is expressed by the individual's cellsand serves as an immunogenic target against which an immune response iselicited. The resulting immune response is broad based: in addition to ahumoral immune response, both arms of the cellular immune response areelicited. The methods of the present invention are useful for conferringprophylactic and therapeutic immunity. Thus, a method of immunizingincludes both methods of protecting an individual from pathogenchallenge, or occurrence or proliferation of specific cells, as well asmethods of treating an individual suffering from pathogen infection,hyperproliferative disease or autoimmune disease. Thus, the presentinvention is useful to elicit broad immune responses against a targetprotein, i.e. proteins specifically associated with pathogens or theindividual's own “abnormal” cells.

The present invention is also useful in combating hyperproliferativediseases and disorders such as cancer, by eliciting an immune responseagainst a target protein that is specifically associated with thehyperproliferative cells. The present invention is further useful incombating autoimmune diseases and disorders by eliciting an immuneresponse against a target protein that is specifically associated withcells involved in the autoimmune condition.

The present invention also provides pharmaceutical kits that comprise acontainer comprising a payload molecule selected from the groupconsisting of a nucleic acid composition, protein composition, smallorganic molecule and mixtures thereof, and a container comprising ayeast cell wall particle and a trapping molecule. Optionally, there isincluded in such kits excipients, carriers, preservatives and vehiclesof the type described above with respect to pharmaceutical compositions.The term pharmaceutical kit is also intended to include multipleinoculants used in the methods of the present invention. Such kitsinclude separate containers comprising different inoculants and transfermoieties. The pharmaceutical kits in accordance with the presentinvention are also contemplated to include a set of inoculants used inthe treatment and immunizing methods and/or therapeutic methods, asdescribed above.

The compositions and methods of the present invention are useful in thefields of both human and veterinary medicine. Accordingly, the presentinvention relates to genetic immunization and therapeutic treatment ofmammals, birds and fish. The methods of the present invention can beparticularly useful for genetic immunization and therapeutic treatmentof mammalian species including human, bovine, ovine, porcine, equine,canine and feline species.

The foregoing and other features and advantages of the particulate drugdelivery system and methods will be apparent from the following moreparticular description of preferred embodiments of the system and methodas illustrated in the accompanying drawings in which like referencecharacters refer to the same parts throughout the different views.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram 100 of a transverse section of a yeastcell wall, showing, from outside to inside, an outer fibrillar layer110, an outer mannoprotein layer 120, a beta glucan layer 130, a betaglucan-chitin layer 140, an inner mannoprotein layer 150, the plasmamembrane 160 and the cytoplasm 170.

FIG. 2A is a diagram of the structure of a yeast cell wall particle;FIG. 2B is a reversed contrast (negative) grayscale image of a colorfluorescence photomicrograph showing staining of the mannan component ofthe yeast cell wall particles by concanavalin-A-FITC (con-A-fluoresceinisothiocyanate, Sigma Chemical, St. Louis, Mo.) showing substantiallycompletely stained yeast cell wall particles 210; FIG. 2C is a diagramof the structure of a YGMP beta glucan-mannan particle, FIG. 2D is areversed contrast (negative) grayscale image of a color fluorescencephotomicrograph showing patchy con-A-FITC staining of a YGMP betaglucan-mannan particle 220; FIG. 2E is a diagram of the structure of aYGP beta glucan particle and FIG. 2F is a reversed contrast (negative)grayscale image of a color fluorescence micrograph showing the absenceof con-A-FITC staining.

FIG. 3A is a reversed contrast (negative) grayscale image of a colorlight photomicrograph of cells exposed to YGP particles loaded withfluorescent labeled pIRES plasmid with PEI as the cationic trappingpolymer and CTAB as a cationic detergent, indicating a cell 310 and FIG.3B is a reversed contrast (negative) grayscale image of a colorfluorescence photomicrograph of the same field of cells showing brightstaining representing fluorescent YGP particles internalized by the samecell 310 indicated in FIG. 3B.

FIG. 4A is a reversed contrast (negative) grayscale image of a colorfluorescence photomicrograph of cells, e.g., an indicated cell 410,exposed to fluorescent labeled YGP particles, FIG. 4B is a reversedcontrast (negative) grayscale image of a color fluorescencephotomicrograph of cells, e.g., an indicated cell 420, exposed to YGPparticles containing pIRES DNA, a cationic trapping polymerpolyethylenimine (PEI) and cationic detergenthexadecyltrimethylammoniumbromide (also known as cetyltrimethylammoniumbromide or CTAB) expressing GFP and FIG. 4C is a reversed contrast(negative) grayscale image of a color fluorescence photomicrograph ofcells, e.g., an indicated cell 430, exposed to YGP particles containingpIRES DNA, a cationic trapping polymer chitosan and cationic detergentCTAB expressing GFP.

FIG. 5A is a reversed contrast (negative) grayscale image of a colorcombined light and fluorescence photomicrograph of cells, e.g., anindicated cell 510, exposed to fluorescent labeled YGP particles; FIG.5B is a graphic representation of the results of a fluorescenceactivated cell sorting (FACS) study showing a major peak 520representing the distribution of signals from cells that haveinternalized fluorescent labeled YGP particles and a minor peak 530representing the distribution of signals from cells without fluorescentlabeled YGP particles; FIG. 5C is a reversed contrast (negative)grayscale image of a color light photomicrograph of cells, e.g., anindicated cell 540, exposed to YGP particles containing fluorescentlabeled DNA, a cationic trapping polymer PEI and cationic detergentCTAB; FIG. 5D is a reversed contrast (negative) grayscale image of acolor fluorescence photomicrograph of the same field of cells showingthe same indicated cell 540, FIG. 5E is a graphic representation of theresults of a FACS study showing a major peak 610 representing thedistribution of signals from cells that have internalized YGP particleswith fluorescent DNA payload and a shoulder 620 representing thedistribution of signals from cells without YGP particles; FIG. 5F is areversed contrast (negative) grayscale image of a color lightphotomicrograph of cells, e.g., an indicated cell 710, incubated withYGP particles containing fluorescent antisense RNA payload; FIG. 5G is areversed contrast (negative) grayscale image of a color fluorescencephotomicrograph of the same field of cells showing the same indicatedcell 710; FIG. 5H is a reversed contrast (negative) grayscale image of acolor light micrograph of cells, e.g., an indicated cell 810, incubatedwith YGP particles containing fluorescent labeled siRNA, PEI and CTABand FIG. 5I is a reversed contrast (negative) grayscale image of a colorfluorescence photomicrograph of the same field of cells showing the sameindicated cell 810 containing internalized YGP particles withfluorescent RNAi payload.

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention.

DETAILED DESCRIPTION OF THE INVENTION

In preferred embodiments, the invention provides a particulate deliverysystem comprising an extracted yeast cell wall particle and at least onepayload trapping molecule. Preferably, the yeast cell wall particle is a2-4 micron yeast cell wall ghost.

Payload Trapping Molecules

The payload trapping molecule is preferably a pharmaceuticallyacceptable excipient. The payload and trapping molecule are both solublein the solvent system; the solvent system must be absorbed through theyeast cell particle carbohydrate matrix allowing the absorption of thepayload and trapping polymer. The payload and trapping molecule arepreferably water soluble. In preferred embodiments, the trappingmolecule is biodegradable.

The mechanism of action of the trapping reaction with a given payloaddictates the choice of payload trapping molecule. For electrostaticinteractions a charged payload trapping molecule of opposite charge ofthe payload is required. For physical entrapment, the payload trappingmolecule suitably participates in the formation of a matrix that reducesthe diffusion of a payload. In other embodiments, the payload trappingmolecule contributes a hydrophobic binding property that contributes tothe retention of the payload. In further embodiments, the payloadtrapping molecule selectively binds to the payload, providing anaffinity interaction that contributes to the retention of the payload.

In general, polyelectrolytes can be suitable payload trapping molecules.Several suitable polyelectrolytes are disclosed in U.S. Pat. No.6,133,229. The polyelectrolyte may be a cationic or anionicpolyelectrolyte. Amphoteric polyelectrolytes may also be employed. Thecationic polyelectrolyte is preferably a polymer with cationic groupsdistributed along the molecular chain. The cationic groups, which incertain embodiments may include quaternary ammonium-derived moieties,may be disposed in side groups pendant from the chain or may beincorporated in it. Examples of cationic polyelectrolytes include:copolymers of vinyl pyrollidone and quaternary methyl methacrylate e.g.,GAFQUAT® series (755N, 734, HS-100) obtained from ISP; substitutedpolyacrylamides; polyethyleneimine, polypropyleneimine and substitutedderivatives; polyamine homopolymers (GOLCHEM® CL118); polyamineco-polymers (e.g., condensates of epichlorohydrin and mono ordimethylamine); polydiallyl dimethyl ammonium chloride (polyDADMAC);substituted dextrans; modified guar gum (substituted withhydroxypropytrimonium chloride); substituted proteins (e.g., quaternarygroups substituted on soya protein and hydrolysed collagen); polyaminoacids (e.g., polylysine); low molecular weight polyamino compounds(e.g., spermine and spermidine). Natural or artificial polymers may beemployed. Cationic polyelectrolytes with MW 150 to 5,000,000, preferably5000 to 500,000, more preferably 5000 to 100,000 may be employed. Anamount of 0.01 to 10% is preferred, more preferably 0.1 to 2% w/v,especially 0.05 to 5%.

The anionic polyelectrolyte is preferably a polymer with anionic groupsdistributed along the molecular chain. The anionic groups, which mayinclude carboxylate, sulfonate, sulphate or other negatively chargedionisable groupings, may be disposed upon groups pendant from the chainor bonded directly to the polymer backbone. Natural or artificialpolymers may be employed.

Examples of anionic polyelectrolytes include: a copolymer of methylvinyl ether and maleic anhydride, a copolymer of methyl vinyl ether andmaleic acid, (Gantrez AN-series and S-series, respectively,International Specialty Products, Wayne, N.J.); alginic acid and salts;carboxymethyl celluloses and salts; substituted polyacrylamides (egsubstituted with carboxylic acid groups); polyacrylic acids and salts;polystyrene sulfonic acids and salts; dextran sulphates; substitutedsaccharides e.g., sucrose octosulfate; heparin. Anionic polyelectrolyteswith MW of 150 to 5,000,000 may be used, preferably 5000 to 500,000,more preferably 5000 to 100,000. An amount of 0.01% to 10% is preferredespecially 0.05 to 5% more especially 0.1 to 2% w/v.

Biological polymers, such as polysaccharides, are preferred trappingpolymers. Preferably, the polymers are processed to an average molecularweight to less than 100,000 Daltons. The polymers are preferablyderivatized to provide cationic or anionic characteristics. Suitablepolysaccharides include chitosan (deacetylated chitin), alginates,dextrans, such as 2-(diethylamino) ethyl ether dextran (DEAE-dextran)and dextran sulphate, xanthans, locust bean gums and guar gums.

Two general classes of cationic molecules are suitable for use astrapping molecules with negatively charged payloads such aspolynucleotides: cationic polymers and cationic lipids.

A wide variety of cationic polymers have been shown to mediate in vitrotransfection, ranging from proteins [such as histones (Fritz, J. D., etal, (1996) Hum. Gene Ther. 7, 1395-1404) and high mobility group (HMG)proteins (Mistry A. R., et al. (1997) BioTechniques 22, 718-729)] andpolypeptides [such as polylysine (Wu, G. Y. & Wu, C. H. (1987) J. Biol.Chem. 262, 4429-4432, Wagner, E., et al., (1991) Bioconjugate Chem. 2,226-231, short synthetic peptides (Gottschalk, S., et al., (1996) GeneTher. 3, 448-457; Wadhwa, M. S., et al., (1997) Bioconjugate Chem. 8,81-88), and helical amphiphilic peptides (Legendre, J. Y., et al.,(1997) Bioconjugate Chem. 8, 57-63; Wyman, T. B., et al., (1997)Biochemistry 36, 3008-3017)] to synthetic polymers [such aspolyethyleneimine (Boussif, O., et al., (1996) Gene Ther. 3, 1074-1080),cationic dendrimers (Tang, M. X., et al., (1996) Bioconjugate Chem. 7,703-714; Haensler, J. et al., (1993) Bioconjugate Chem. 4, 372-379), andglucaramide polymers (Goldman, C. K., et al., (1997) Nat. Biotech. 15,462-466)]. Other suitable cationic polymers include N-substitutedglycine oligomers (peptoids) (Murphy, J. E., et al, A combinatorialapproach to the discovery of efficient cationic peptoid reagents forgene delivery, Proc Natl Acad. Sci. USA, 1998 95 (4)1517-1522),poly(2-methyl-acrylic acid2-[(2-dimethylamino)-ethyl)-methyl-amino]-ethyl ester), abbreviated aspDAMA, and poly(2-dimethylamino ethyl)-methacrylate (pDMAEMA) (Funhoff,A. M., et al., 2004 Biomacromolecules, 5, 32-39).

Cationic lipids are also known in the art to be suitable fortransfection. Felgner, P.L1, et al., Lipofection: a highly efficient,lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci USA. 198784(21):7413-7. Suitable cationic lipids includeN-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),[N,N,N′,N′-tetramethyl-N,N′-bis(2-hydroxyethyl)-2,3-di(oleoyloxy)-1,4-butanediammoniumiodide](Promega Madison, Wis., USA), dioctadecylamidoglycyl spermine(Promega Madison, Wis., USA),N-[1-(2,3-Dioleoyloxy)]-N,N,N-trimethylammonium propane methylsulfate(DOTAP), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride,1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide(DMRIE), dimyristoleoyl phosphonomethyl trimethyl ammonium (DMPTA) (seeFloch et al. 1997. Cationic phosphonolipids as non-viral vectors for DNAtransfection in hematopoietic cell lines and CD34+ cells. Blood Cells,Molec. & Diseases 23: 69-87),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl),ammonium salt (Avanti Polar Lipids, Inc. Alabaster, Ala., US),1,2-dioleoyl-3-trimethylammonium-propane chloride (Avanti Polar Lipids,Inc. Alabaster, Ala., US), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine(Avanti Polar Lipids, Inc. Alabaster, Ala., US) and1,3-dioleoyloxy-2-(6-carboxyspermyl)propylamide (DOSPER).

Polyamines suitable as cationic trapping molecules are described in U.S.Pat. Nos. 6,379,965 and 6,372,499.

Payload Molecules

The particulate delivery system of the present invention is useful forin vivo or in vitro delivery of payload molecules including, but limitedto, polynucleotides such as oligonucleotides, antisense constructs,siRNA, enzymatic RNA, and recombinant DNA constructs, includingexpression vectors.

In other preferred embodiments, the particulate delivery system of thepresent invention is useful for in vivo or in vitro delivery of payloadmolecules such as amino acids, peptides and proteins. By “protein” ismeant a sequence of amino acids for which the chain length is sufficientto produce the higher levels of tertiary and/or quaternary structure.This is to distinguish from “peptides” or other small molecular weightdrugs that do not have such structure. Typically, the protein hereinwill have a molecular weight of at least about 15-20 kD, preferably atleast about 20 kD.

Examples of proteins encompassed within the definition herein includemammalian proteins, such as, e.g., growth hormone (GH), including humangrowth hormone, bovine growth hormone, and other members of the GHsupergene family; growth hormone releasing factor, parathyroid hormone;thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin; insulinA-chain; insulin B-chain; proinsulin; follicle stimulating hormone;calcitonin; luteinizing hormone; glucagon; clotting factors such asfactor VIIIC, factor IX tissue factor, and von Willebrands factor;anti-clotting factors such as Protein C; atrial natriuretic factor; lungsurfactant; a plasminogen activator, such as urokinase or tissue-typeplasminogen activator (t-PA); bombazine; thrombin; alpha tumor necrosisfactor, beta tumor necrosis factor; enkephalinase; RANTES (regulated onactivation normally T-cell expressed and secreted); human macrophageinflammatory protein (MIP-1-alpha); serum albumin such as human serumalbumin; mullerian-inhibiting substance; relaxin A-chain; relaxinB-chain; prorelaxin; mouse gonadotropin-associated peptide; DNase;inhibin; activin; vascular endothelial growth factor (VEGF); receptorsfor hormones or growth factors; an integrin; protein A or D; rheumatoidfactors; a neurotrophic factor such as bone-derived neurotrophic factor(BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or anerve growth factor such as NGF-beta; platelet-derived growth factor(PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growthfactor (EGF); transforming growth factor (TGF) such as TGF-alpha andTGF-beta, including TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, orTGF-beta5; insulin-like growth factor-I and -II (IGF-I and IGF-II);des(1-3)-IGF-I (brain IGF-D; insulin-like growth factor bindingproteins; CD proteins such as CD3, CD4, CD8, CD19 and CD20;osteoinductive factors; immunotoxins; a bone morphogenetic protein(BMP); T-cell receptors; surface membrane proteins; decay acceleratingfactor (DAF); a viral antigen such as, for example, a portion of theAIDS envelope; transport proteins; homing receptors; addressins;regulatory proteins; immunoadhesins; antibodies; and biologically activefragments or variants of any of the above-listed polypeptides.

The members of the OH supergene family include growth hormone,prolactin, placental lactogen, erythropoietin, thrombopoietin,interleukin-2, interleukin-3, interleukin-4, interleukin-5,interleukin-6, interleukin-7, interleukin-9, interleukin-10,interleukin-11, interleukin-12 (p35 subunit), interleukin-13,interleukin-15, oncostatin M, ciliary neurotrophic factor, leukemiainhibitory factor, alpha interferon, beta interferon, gamma interferon,omega interferon, tau interferon, granulocyte-colony stimulating factor,granulocyte-macrophage colony stimulating factor, macrophage colonystimulating factor, cardiotrophin-1 and other proteins identified andclassified as members of the family.

The protein payload molecule is preferably essentially pure anddesirably essentially homogeneous (i.e. free from contaminating proteinsetc). “Essentially pure” protein means a composition comprising at leastabout 90% by weight of the protein, based on total weight of thecomposition, preferably at least about 95% by weight. “Essentiallyhomogeneous” protein means a composition comprising at least about 99%by weight of protein, based on total weight of the composition. Proteinsmay be derived from naturally occurring sources or produced byrecombinant technology. Proteins include protein variants produced byamino acid substitutions or by directed protein evolution (Kurtzman, A.L., et al., Advances in directed protein evolution by recursive geneticrecombination: applications to therapeutic proteins, Curr OpinBiotechnol. 2001 12(4): 361-70) as well as derivatives, such asPEGylated proteins.

In certain embodiments, the protein is an antibody. The antibody maybind to any of the above-mentioned molecules, for example. Exemplarymolecular targets for antibodies encompassed by the present inventioninclude CD proteins such as CD3, CD4, CD8, CD19, CD20 and CD34; membersof the HER receptor family such as the EGF receptor, HER2, HER3 or HER4receptor; cell adhesion molecules such as LFA-1, Mol, p150,95, VLA-4,ICAM-1, VCAM and alphav/beta3 integrin including either alpha or betasubunits thereof (e.g. anti-CD11a, anti-CD18 or anti-CD11b antibodies);growth factors such as VEGF; IgE; blood group antigens; flk2/flt3receptor; obesity (OB) receptor; protein C, etc.

In addition to peptides, polypeptides and polynucleotides, theparticulate delivery system of the present invention is suitable for thedelivery of smaller molecules, preferably for the delivery ofpharmaceutically active agent, more preferably therapeutic smallmolecules. Suitable small molecule payloads for the delivery system ofthe present invention include contraceptive agents such as diethylstilbestrol, 17-beta-estradiol, estrone, ethinyl estradiol, mestranol,and the like; progestins such as norethindrone, norgestryl, ethynodioldiacetate, lynestrenol, medroxyprogesterone acetate, dimethisterone,megestrol acetate, chlormadinone acetate, norgestimate, norethisterone,ethisterone, melengestrol, norethynodrel and the like; and spermicidalcompounds such as nonylphenoxypolyoxyethylene glycol, benzethoniumchloride, chlorindanol and the like. Preferably, for such steroidalpayloads, a mixture of trapping molecules is used, comprising asufficient amount of a detergent to solubilize the payload and a polymerto retain the payload within the yeast cell wall particle.

Other active agents that can be incorporated in the delivery system ofthe present invention include gastrointestinal therapeutic agents suchas aluminum hydroxide, calcium carbonate, magnesium carbonate, sodiumcarbonate and the like; non-steroidal antifertility agents;parasympathomimetic agents; psychotherapeutic agents; majortranquilizers such as chloropromazine HCl, clozapine, mesoridazine,metiapine, reserpine, thioridazine and the like; minor tranquilizerssuch as chlordiazepoxide, diazepam, meprobamate, temazepam and the like;rhinological decongestants; sedative-hypnotics such as codeine,phenobarbital, sodium pentobarbital, sodium secobarbital and the like;other steroids such as testosterone and testosterone propionate;sulfonamides; sympathomimetic agents; vaccines; vitamins and nutrientssuch as the essential amino acids, essential fats and the like;antimalarials such as 4-aminoquinolines, 8-aminoquinolines,pyrimethamine and the like; anti-migraine agents such as mazindol,phentermine and the like; anti-Parkinson agents such as L-dopa;anti-spasmodics such as atropine, methscopolamine bromide and the like;antispasmodics and anticholinergic agents such as bile therapy,digestants, enzymes and the like; antitussives such as dextromethorphan,noscapine and the like; bronchodilators; cardiovascular agents such asanti-hypertensive compounds, Rauwolfia alkaloids, coronary vasodilators,nitroglycerin, organic nitrates, pentaerythritotetranitrate and thelike; electrolyte replacements such as potassium chloride;ergotalkaloids such as ergotamine with and without caffeine,hydrogenated ergot alkaloids, dihydroergocristine methanesulfate,dihydroergocomine methanesulfonate, dihydroergokroyptine methanesulfateand combinations thereof; alkaloids such as atropine sulfate,Belladonna, hyoscine hydrobromide and the like; analgesics; narcoticssuch as codeine, dihydrocodienone, meperidine, morphine and the like;non-narcotics such as salicylates, aspirin, acetaminophen,d-propoxyphene and the like.

In preferred embodiments, the system of the present invention is used todeliver antibiotics such as the cephalosporins, chloramphenical,gentamicin, kanamycin A, kanamycin B, the penicillins, ampicillin,streptomycin A, antimycin A, chloropamtheniol, metronidazole,oxytetracycline penicillin G, the tetracyclines, and the like. Inpreferred embodiments, the ability of the body's macrophages toinactivate pathogens is enhanced by the delivery of antibiotics, such astetracycline, to the macrophages.

In other preferred embodiments, the present invention provides a systemto deliver anti-cancer agents; anti-convulsants such as mephenyloin,phenobarbital, trimethadione; anti-emetics such as thiethylperazine;antihistamines such as chlorophinazine, dimenhydrinate, diphenhydramine,perphenazine, tripelennamine and the like; anti-inflammatory agents suchas hormonal agents, hydrocortisone, prednisolone, prednisone,non-hormonal agents, allopurinol, aspirin, indomethacin, phenylbutazoneand the like; prostaglandins; cytotoxic drugs such as thiotepa,chlorambucil, cyclophosphamide, melphalan, nitrogen mustard,methotrexate and the like.

Vaccines

In preferred embodiments, the particulate delivery system of the presentinvention is useful in providing oral delivery of vaccines. In preferredembodiments, the system is used to deliver antigens, such as antigens ofsuch microorganisms as Neisseria gonorrhea, Mycobacterium tuberculosis,Herpes virus (humonis, types 1 and 2), Candida albicans, Candidatropicalis, Trichomonas vaginalis, Haemophilus vaginalis, Group BStreptococcus sp., Microplasma hominis, Hemophtlus ducreyi, Granulomainguinale, Lymphopathia venereum, Treponema pallidum, Brucella abortus,Brucella melitensis, Brucella suis, Brucella cents, Campylobacter fetus,Campylobacrer fetus intestinalis, Leptospira pomona, Listeriamonocytogenes, Brucella ovis, equine herpes virus 1, equine arteritisvirus, IBR-IBP virus, BVD-MB virus, Chlamydia psittaci, Trichomonasfoetus, Toxoplasma gondii, Escherichia coli, Actinobacillus equuli,Salmonella aborrus ovis, Salmonella abortus equi, Pseudomonasaeruginosa, Corynebacterium equi, Corynebacterium pyogenes,Actinobaccilus seminis, Mycoplasma bovigenitalium, Aspergillusfumigatus, Absidia ramosa, Trypanosoma equiperdum, Babesia caballi,Clostridium tetani, Clostridium botulinum and the like. In otherembodiments, the system can be used to deliver neutralizing antibodiesthat counteract the above microorganisms.

In other embodiments, the system can be used to deliver enzymes such asribonuclease, neuramidinase, trypsin, glycogen phosphorylase, spermlactic dehydrogenase, sperm hyaluronidase, adenossinetriphosphatase,alkaline phosphatase, alkaline phosphatase esterase, amino peptidase,trypsin chymotrypsin, amylase, muramidase, acrosomal proteinase,diesterase, glutamic acid dehydrogenase, succinic acid dehydrogenase,beta-glycophosphatase, lipase, ATP-ase alpha-peptategamma-glutamylotranspeptidase, sterol-3-beta-ol-dehydrogenase,DPN-di-aprorase.

In preferred embodiments, the system can deliver antigens ofbioterrorism critical biological agents, including Category A agentssuch as variola major (smallpox), Bacillus anthracis (anthrax), Yersiniapestis (plague), Clostridium botulinum toxin (botulism), Francisellatularensis (tularaemia), filoviruses (Ebola hemorrhagic fever, Marburghemorrhagic fever), arenaviruses (Lassa (Lassa fever), Junin (Argentinehemorrhagic fever) and related viruses); Category B agents such asCoxiella burnetti (Q fever), Brucella species (brucellosis),Burkholderia mallei (glanders), alphaviruses (Venezuelanencephalomyelitis, eastern & western equine encephalomyelitis), ricintoxin from Ricinus communis (castor beans), epsilon toxin of Clostridiumperfringens; Staphylococcus enterotoxin B, Salmonella species, Shigelladysenteriae, Escherichia coli strain O157:H7, Vibrio cholerae,Cryptosporidium parvum; and Category C agents such as nipah virus,hantaviruses, tickborne hemorrhagic fever viruses, tickborneencephalitis viruses, yellow fever, and multidrug-resistanttuberculosis.

In preferred embodiments, the system can be used to deliver inactivatedantigenic toxins, such as anatoxin antigens, including toxoids(inactivated but antigenic toxins), and toxoid conjugates. In preferredembodiments, the toxoid is an inactivated microbial toxin. In otherembodiments, the toxoid is an inactivated plant toxin. In furtherembodiments, the toxoid is an inactivated animal toxin. In certainembodiments, the system can be used to deliver toxoids such as pertussistoxoid, Corynebacterium diphtheriae toxoid, tetanus toxoid, Haemophilusinfluenzae type b-tetanus toxoid conjugate, Clostridium botulinum Dtoxoid, Clostridium borulinum E toxoid, toxoid produced from Toxin A ofClostridium difficile, Vibrio cholerae toxoid, Clostridium perfringensTypes C and D toxoid, Clostridium chauvoei toxoid, Clostridium novyi(Type B) toxoid, Clostridium septicum toxoid, recombinant HIV tat IIIBtoxoid, Staphylococcus toxoid, Actinobacillus pleuropneumoniae Apx Itoxoid, Actinobacillus pleuropneumoniae Apx II toxoid, Actinobacilluspleuropneumoniae Apx III toxoid, Actinobacillus pleuropneumoniae outermembrane protein (OMP) toxoid, Pseudomonas aeruginosa elastase toxoid,snake venom toxoid, ricin toxoid, Mannheimia haemolytica toxoid,Pasteurella multocida toxoid, Salmonella typhimurium toxoid, Pasteurellamultocida toxoid, and Bordetella bronchiseptica toxoid.

Techniques of making a toxoid from a corresponding toxin, e.g. chemicaltreatment with formaldehyde or aluminum salts or gamma irradiation, areknown in the art. Recombinant methods of converting a toxin to a toxoidare also known (Fromen-Romano, C., et al., Transformation of anon-enzymatic toxin into a toxoid by genetic engineering, ProteinEngineering vol. 10 no. 10 pp. 1213-1220, 1997). In preferredembodiments, the system of the present invention can be used to delivera recombinant toxoid. In other preferred embodiments, the system of thepresent invention can be used to deliver an expression vector encoding arecombinant toxoid.

In order to produce a genetic vaccine to protect against pathogeninfection, genetic material which encodes immunogenic proteins againstwhich a protective immune response can be mounted, must be included inthe nucleic acid composition. Whether the pathogen infectsintracellularly, for which the present invention is particularly useful,or extracellularly, it is unlikely that all pathogen antigens willelicit a protective response. Because DNA and RNA are both relativelysmall and can be produced relatively easily, the present inventionprovides the additional advantage of allowing for vaccination withmultiple pathogen antigens. The nucleic acid composition used in thegenetic vaccine can include genetic material that encodes many pathogenantigens. For example, several viral genes may be included in a singleconstruct, thereby providing multiple targets. In addition, multipleinoculants which can be delivered to different cells in an individualcan be prepared to collectively include, in some cases, a complete or,more preferably, an incomplete, e.g., nearly complete set of genes inthe vaccine. For example, a complete set of viral genes may beadministered using two constructs which each contain a different half ofthe genome which are administered at different sites. Thus, an immuneresponse may be invoked against each antigen without the risk of aninfectious virus being assembled. This allows for the introduction ofmore than a single antigen target and can eliminate the requirement thatprotective antigens be identified.

In accordance with the present invention there is also provided a methodof conferring a broad based protective immune response againsthyperproliferating cells that are characteristic of hyperproliferativediseases, as well as a method of treating individuals suffering fromhyperproliferative diseases. As used herein, the term“hyperproliferative diseases” is meant to refer to those diseases anddisorders characterized by hyperproliferation of cells. Examples ofhyperproliferative diseases include all forms of cancer and psoriasis.

It has been discovered that introduction of a nucleic acid compositionthat includes a nucleotide sequence which encodes an immunogenic“hyperproliferating cell”-associated protein into the cells of anindividual, results in the production of those proteins in thevaccinated cells of an individual. As used herein, the term“hyperproliferative-associated protein” is meant to refer to proteinsthat are associated with a hyperproliferative disease. To immunizeagainst hyperproliferative diseases, a nucleic acid composition thatincludes a nucleotide sequence which encodes a protein that isassociated with a hyperproliferative disease is administered to anindividual.

In order for the hyperproliferative-associated protein to be aneffective immunogenic target, it must be a protein that is producedexclusively or at higher levels in hyperproliferative cells as comparedto normal cells. Target antigens include such proteins, fragmentsthereof and peptides which comprise at least an epitope found on suchproteins. In some cases, a hyperproliferative-associated protein is theproduct of a mutation of a gene that encodes a protein. The mutated geneencodes a protein which is nearly identical to the normal protein exceptit has a slightly different amino acid sequence which results in adifferent epitope not found on the normal protein. Such target proteinsinclude those which are proteins encoded by oncogenes such as myb, myc,fyn, and the translocation genes bcr/abl, ras, src, P53, neu, trk andEGRF. In addition to oncogene products as target antigens, targetproteins for anti-cancer treatments and protective regimens includevariable regions of antibodies made by B cell lymphomas, and variableregions of T cell receptors of T cell lymphomas which, in someembodiments, are also used as target antigens for autoimmune diseases.Other tumor-associated proteins can be used as target proteins, such asproteins which are found at higher levels in tumor cells, including theprotein recognized by monoclonal antibody 17-1A and folate bindingproteins.

While the present invention may be used to immunize an individualagainst one or more of several forms of cancer, the present invention isparticularly useful to prophylactically immunize an individual who ispredisposed to develop a particular cancer or who has had cancer and istherefore susceptible to a relapse. Developments in genetics andbiotechnology, as well as epidemiology, allow for the determination ofprobability and risk assessment for the development of cancer in anindividual. Using genetic screening and/or family health histories, itis possible to predict the probability that a particular individual hasfor developing any one of several types of cancer.

Similarly, those individuals who have already developed cancer and whohave been treated to remove the cancer, or are otherwise in remission,are particularly susceptible to relapse and reoccurrence. As part of atreatment regimen, such individuals can be immunized against the cancerthat they have been diagnosed as having had in order to combat such arecurrence. Thus, once it is known that individuals have had a type ofcancer and are at risk of a relapse, they can be immunized in order toprepare their immune systems to combat any future appearance of thecancer.

The present invention also provides a method of treating individualssuffering from hyperproliferative diseases. In such methods, theintroduction of peptide, protein, carbohydrate or nucleic acidcompositions and combinations thereof serves as an immunotherapeutic,directing and promoting the immune system of the individual to combathyperproliferative cells that produce the target protein.

The present invention provides a method of treating individualssuffering from autoimmune diseases and disorders by conferring a broadbased protective immune response against targets that are associatedwith autoimmunity, including cell receptors and cells which produce“self”-directed antibodies.

T cell mediated autoimmune diseases include Rheumatoid arthritis (RA),multiple sclerosis (MS), Sjogren's syndrome, sarcoidosis, insulindependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactivearthritis, ankylosing spondylitis, scleroderma, polymyositis,dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis,Crohn's disease and ulcerative colitis. Each of these diseases ischaracterized by T cell receptors that bind to endogenous antigens andinitiate the inflammatory cascade associated with autoimmune diseases.Vaccination against the variable region of the T cells would elicit animmune response including CTLs to eliminate those T cells.

In RA, several specific variable regions of T cell receptors (TCRs)which are involved in the disease have been characterized. These TCRsinclude Vβ-3, Vβ-14, Vβ-17 and Vα-17. Thus, vaccination with acomposition composed of peptide, protein, carbohydrate or nucleic acidcompositions and combinations thereof that delivers or encodes at leastone of these proteins will elicit an immune response that will target Tcells involved in RA. See: Howell, M. D., et al., 1991 Proc. Natl. Acad.Sci. USA 88:10921-10925; Paliard, X., et al., 1991 Science 253:325-329;Williams, W. V., et al., 1992 J. Clin. Invest. 90:326-333; each of whichis incorporated herein by reference.

In MS, several specific variable regions of TCRs which are involved inthe disease have been characterized. These TCRs include Vβ-7 and Vα-10.Thus, vaccination with a composition composed of peptide, protein,carbohydrate or nucleic acid compositions and combinations thereof thatdelivers or encodes at least one of these proteins will elicit an immuneresponse that will target T cells involved in MS. See: Wucherpfennig, K.W., et al., 1990 Science 248:1016-1019; Oksenberg, J. R., et al., 1990Nature 345:344-346; each of which is incorporated herein by reference.

In scleroderma, several specific variable regions of TCRs which areinvolved in the disease have been characterized. These TCRs includeV-β-6, Vβ-8, Vβ-14 and Vα-16, Vα-3C, Vα-7, Vα-14, Vα-15, Vα-16, Vα-28and Vα-12. Thus, vaccination with a composition composed of peptide,protein, carbohydrate or nucleic acid compositions and combinationsthereof that delivers or encodes for at least one of these proteins willelicit an immune response that will target T cells involved inscleroderma.

In order to treat patients suffering from a T cell mediated autoimmunedisease, particularly those for which the variable region of the TCR hasyet to be characterized, a synovial biopsy can be performed. Samples ofthe T cells present can be taken and the variable region of those TCRsidentified using standard techniques. Vaccines can be prepared usingthis information.

B cell mediated autoimmune diseases include Lupus (SLE), Grave'sdisease, myasthenia gravis, autoimmune hemolytic anemia, autoimmunethrombocytopenia, asthma, cryoglobulinemia, primary biliary sclerosisand pernicious anemia. Each of these diseases is characterized byantibodies which bind to endogenous antigens and initiate theinflammatory cascade associated with autoimmune diseases. Vaccinationagainst the variable region of such antibodies would elicit an immuneresponse including CTLs to eliminate those B cells that produce theantibody.

In order to treat patients suffering from a B cell mediated autoimmunedisease, the variable region of the antibodies involved in theautoimmune activity must be identified. A biopsy can be performed andsamples of the antibodies present at a site of inflammation can betaken. The variable region of those antibodies can be identified usingstandard techniques. Vaccines can be prepared using this information.

In the case of SLE, one antigen is believed to be DNA. Thus, in patientsto be immunized against SLE, their sera can be screened for anti-DNAantibodies and a vaccine can be prepared which includes nucleic acidcompositions that encode the variable region of such anti-DNA antibodiesfound in the sera.

Common structural features among the variable regions of both TCRs andantibodies are well known. The DNA sequence encoding a particular TCR orantibody can generally be found following well known methods such asthose described in Kabat, et al. 1987 Sequence of Proteins ofImmunological Interest U.S. Department of Health and Human Services,Bethesda Md., which is incorporated herein by reference. In addition, ageneral method for cloning functional variable regions from antibodiescan be found in Chaudhary, V. K., et al., 1990 Proc. Natl. Acad. Sci.USA 87:1066, which is incorporated herein by reference.

Gene Therapy

In preferred embodiments, the present invention provides compositionsand methods for the treatment of genetic disorders or conditions havinga genetic component. In further preferred embodiments, the presentinvention provides compositions useful for the manufacture ofpharmaceutical products for the treatment of genetic disorders orconditions having a genetic component.

The Human Genome Project has increased our knowledge of the geneticbasis of disease. See, generally,http:/www.ornl.gov/sci/techresources/Human_Genome/medicine/assist.shtml.

Both environmental and genetic factors have roles in the development ofany disease. A genetic disorder is a disease caused by abnormalities inan individual's genetic material (genome). There are four differenttypes of genetic disorders: (1) single-gene, (2) multifactorial, (3)chromosomal, and (4) mitochondrial.

(1) Single-gene (also called Mendelian or monogenic)—This type is causedby changes or mutations that occur in the DNA sequence of one gene.Genes code for proteins, the molecules that carry out most of the work,perform most life functions, and even make up the majority of cellularstructures. When a gene is mutated so that its protein product can nolonger carry out its normal function, a disorder can result. There aremore than 6,000 known single-gene disorders, which occur in about 1 outof every 200 births. Some examples are cystic fibrosis, sickle cellanemia, Marfan syndrome, Huntington's disease, and hereditaryhemochromatosis.

(2) Multifactorial (also called complex or polygenic)—This type iscaused by a combination of environmental factors and mutations inmultiple genes. For example, different genes that influence breastcancer susceptibility have been found on chromosomes 6, 11, 13, 14, 15,17, and 22. Its more complicated nature makes it much more difficult toanalyze than single-gene or chromosomal disorders. Some of the mostcommon chronic disorders are multifactorial disorders. Examples includeheart disease, high blood pressure, Alzheimer's disease, arthritis,diabetes, cancer, and obesity. Multifactorial inheritance also isassociated with heritable traits such as fingerprint patterns, height,eye color, and skin color.

(3) Chromosomal—Chromosomes, distinct structures made up of DNA andprotein, are located in the nucleus of each cell. Because chromosomesare carriers of genetic material, such abnormalities in chromosomestructure as missing or extra copies or gross breaks and rejoinings(translocations), can result in disease. Some types of major chromosomalabnormalities can be detected by microscopic examination. Down syndromeor trisomy 21 is a common disorder that occurs when a person has threecopies of chromosome 21.

(4) Mitochondrial—This relatively rare type of genetic disorder iscaused by mutations in the nonchromosomal DNA of mitochondria.Mitochondria are small round or rod-like organelles that are involved incellular respiration and found in the cytoplasm of plant and animalcells. Each mitochondrion may contain 5 to 10 circular pieces of DNA.

In preferred embodiments, the particulate delivery system of the presentinvention is used to administer at least one polynucleotide comprising acompensating gene. In other preferred embodiments, the particulatedelivery system of the present invention is used to administer at leastone polynucleotide encoding a gene product of a missing gene, whereinthe expression of the gene product is useful in the treatment of thegenetic disorder or the genetic component of a condition. In preferredembodiments, the particulate delivery system of the present inventionincluding the desired payload molecule is useful for the manufacture ofa pharmaceutical product for the treatment of genetic disorder or thegenetic component of a condition. Such pharmaceutical products aresuitably administered orally, rectally, parenterally, (for example,intravenously, intramuscularly, or subcutaneously) intracisternally,intravaginally, intraperitoneally, intravesically, locally (for example,powders, ointments or drops), or as a buccal or nasal spray. Thepharmaceutical products are preferably administered orally, buccally,and parenterally, more preferably orally. Particles loaded withdifferent payloads, e.g. a polynucleotide, a polynucleotide expressionvector or a small molecule therapeutic can be mixed in the appropriateproportions and administered together, e.g., in a capsule, forcombination therapy.

In aspects of the present invention that relate to gene therapy, thenucleic acid compositions contain either compensating genes or genesthat encode therapeutic proteins. Examples of compensating genes includea gene that encodes dystrophin or a functional fragment, a gene tocompensate for the defective gene in patients suffering from cysticfibrosis, a gene to compensate for the defective gene in patientssuffering from ADA, and a gene encoding Factor VIII. Examples of genesencoding therapeutic proteins include genes which encodeserythropoietin, interferon, LDL receptor, GM-CSF, IL-2, IL-4 and TNF.Additionally, nucleic acid compositions which encode single chainantibody components which specifically bind to toxic substances can beadministered. In some preferred embodiments, the dystrophin gene isprovided as part of a mini-gene and used to treat individuals sufferingfrom muscular dystrophy. In some preferred embodiments, a mini-genewhich contains coding sequence for a partial dystrophin protein isprovided. Dystrophin abnormalities are responsible for both the milderBecker's Muscular Dystrophy (BMD) and the severe Duchenne's MuscularDystrophy (DMD). In BMD dystrophin is made, but it is abnormal in eithersize and/or amount. The patient is mild to moderately weak. In DMD noprotein is made and the patient is wheelchair-bound by age 13 andusually dies by age 20. In some patients, particularly those sufferingfrom BMD, partial dystrophin protein produced by expression of amini-gene delivered according to the present invention can provideimproved muscle function.

In preferred embodiments, the particulate delivery system of the presentinvention provides compositions and methods for the treatment of geneticdisorders and conditions believed to have a genetic component, such asAarskog-Scott syndrome, Aase syndrome, achondroplasia, acrodysostosis,addiction, adreno-leukodystrophy, albinism, ablepharon-macrostomiasyndrome, alagille syndrome, alkaptonuria, alpha-1 antitrypsindeficiency, Alport's syndrome, Alzheimer's disease, asthma, autoimmunepolyglandular syndrome, androgen insensitivity syndrome, Angelmansyndrome, ataxia, ataxia telangiectasia, atherosclerosis, attentiondeficit hyperactivity disorder (ADHD), autism, baldness, Batten disease,Beckwith-Wiedemann syndrome, Best disease, bipolar disorder,brachydactyl), breast cancer, Burkitt lymphoma, chronic myeloidleukemia, Charcot-Marie-Tooth disease, Crohn's disease, cleft lip,Cockayne syndrome, Coffin Lowry Syndrome, colon cancer, congenitaladrenal hyperplasia (CAH), Cornelia de Lange Syndrome, CostelloSyndrome, Cowden Syndrome, Craniofrontonasal Dysplasia, Crigler-NajjarSyndrome, Creutzfeldt-Jakob Disease (CJD), cystic fibrosis, deafness,depression, diabetes, diastrophic dysplasia, DiGeorge Syndrome, Down'sSyndrome, dyslexia, Duchenne muscular dystrophy, Dubowitz Syndrome,ectodermal dysplasia, Ellis-van Creveld syndrome, Ehlers-Danlos,Epidermolysis Bullosa (EB), epilepsy, essential tremor, familialhypercholesterolemia, familial Mediterranean fever, Fragile X Syndrome,Friedreich's ataxia, Gaucher disease, glaucoma, glucose galactosemalabsorption, glutaricaciduria, gyrate atrophy, Goldberg ShprintzenSyndrome (velocardiofacial syndrome), Gorlin Syndrome, Hailey-HaileyDisease, hemihypertrophy, hemochromatosis, hemophilia, hereditary motorand sensory neuropathy (HMSN), hereditary non polyposis colorectalcancer (HNPCC), Huntington's disease, immunodeficiency with hyper-IgM,juvenile onset diabetes, Klinefelter's Syndrome, Kabuki Syndrome,Leigh's Disease (or Syndrome) Long QT Syndrome, lung cancer, malignantmelanoma, manic depression, Marfan Syndrome, Menkes syndrome,miscarriage, mucopolysaccharide disease, multiple endocrine neoplasia,multiple sclerosis, muscular dystrophy, myotrophic lateral sclerosis,myotonic dystrophy, neurofibromatosis, Niemann-Pick disease, NoonanSyndrome, obesity, ovarian cancer, p53 tumor suppressor, pancreaticcancer, Parkinson disease, paroxysmal nocturnal hemoglobinuria, Pendredsyndrome, peroneal muscular atrophy, phenylketonuria (PKU), polycystickidney disease, Prader-Willi Syndrome, primary biliary cirrhosis,prostate cancer, REAR Syndrome, Refsum disease, retinitis pigmentosa,retinoblastoma, Rett Syndrome, Sanfilippo Syndrome, schizophrenia,severe combined immunodeficiency, sickle cell anemia, spina bifida,spinal muscular atrophy, spinocerebellar atrophy, SRY: sexdetermination, Sudden Adult Death Syndrome, Tangier disease, Tay-Sachsdisease, thrombocytopenia absent radius syndrome, Townes-BrocksSyndrome, tuberous sclerosis, Turner syndrome, Usher syndrome, vonHippel-Lindau syndrome, Waardenburg syndrome, Weaver syndrome, Wernersyndrome, Williams syndrome, Wilson's Disease, xeroderma pigmentosum andZellweger syndrome.

In other preferred embodiments, the particulate delivery system of thepresent invention provides compositions and methods for the treatment ofgenetic disorders and conditions believed to have a genetic componentthat are manifested as metabolic disorders, such as protein-relateddisorders, including Sickle-Cell Anemia and beta-Thalassemias,alpha-Thalassemias, Marfan's Syndrome, Ehlers-Danlos Type I,Ehlers-Danlos Type II, Ehlers-Danlos Type m, Ehlers-Danlos Type IVautosomal dominant, Ehlers-Danlos Type IV autosomal recessive,Ehlers-Danlos Type IV-D, Ehlers-Danlos Type V, Ehlers-Danlos Type VI,Ehlers-Danlos Type VII autosomal dominant, Ehlers-Danlos Type VIIautosomal recessive, Ehlers-Danlos Type VIII. Ehlers-Danlos withPlatelet Dysfunction, Cutis Laxa, Cutis Laxa recessive Type I, OccipitalHorn Syndrome Cutis Laxa, X-linked, Osteogenesis Imperfecta Type I,Osteogenesis Imperfecta Type I-C, Osteogenesis Imperfecta Silent TypeII/II, Osteogenesis Imperfecta Type IV, Osteogenesis Imperfecta NeonatalLethal form, and Osteogenesis Imperfecta progressively deforming.

In further preferred embodiments, the particulate delivery system of thepresent invention provides compositions and methods for the treatment ofgenetic disorders of the clotting system, such as afibrinogenemia,complete loss of fibrinogen, Factor I; dysfibrinogenemia dysfunctionalfibrinogen, Factor I; Factor II disorders; tissue factor deficiency;Factor V deficiency, labile Factor deficiency, Factor VII deficiency,Factor VIII deficiency (Hemophilia A), Factor IX deficiency (HemophiliaB), Factor X deficiency, Factor XI deficiency, Rosenthal Syndrome,Plasma Thromboplastin Antecedent (PTA) deficiency, Factor XIIdeficiency, Hageman factor deficiency, Factor XIII deficiency, Factor V& VIII Combined deficiency, Factor VIII & IX combined deficiency, FactorIX & XI Combined deficiency, Protein C deficiency, Protein S deficiency,thrombophilia, antithrombin III deficiency, giant platelet syndrome,platelet glycoprotein Ib deficiency, von Willebrand disease, FletcherFactor deficiency and prekallikrein deficiency.

In further preferred embodiments, the particulate delivery system of thepresent invention provides compositions and methods for the treatment ofglycogen storage disorders, such as Type 0, Type I (von Gierke'sdisease), Type Ib, Type Ic, Type II (Pompe disease), Type IIb (Danondisease), Type III (Cori disease or Forbes disease), Type IV (Andersendisease), Type V (McArdle disease), Type VI (Hers disease), Type VII(Tarui disease), Type VIII, Type IX, and Type XI (Fanconi-Bickelsyndrome).

In yet further preferred embodiments, the particulate delivery system ofthe present invention provides compositions and methods for thetreatment of defects in fructose, galactose and glycerol metabolism,such as hereditary fructose intolerance, aldolase B deficiency;fructosuria, hepatic fructokinase deficiency; classic galactosemia,galactose epimerase deficiency; galactokinase deficiency;hyperglycerolemia and glycerol kinase deficiency.

In yet further preferred embodiments, the particulate delivery system ofthe present invention provides compositions and methods for thetreatment of defects in cholesterol and lipoprotein metabolism, such asapolipoprotein(a)—Lp(a), hyperlipoproteinemia Type I;hyperlipoproteinemia Type Ib; apolipoprotein C-II deficiency;hyperlipoproteinemia Type Ic, chylomicronemia; familialhypercholesterolemia, Type II hyperlipoproteinemia; hyperlipoproteinemiaType II, familial hyperbetalipoproteinemia; hyperlipoproteinemia TypeIII, apolipoprotein E deficiency; hyperlipoproteinemia Type IV;hyperlipoproteinemia Type V; familial LCAT deficiency; Wolman disease;lipoprotein lipase deficiency; familial hypertriglyceridemia;hyperlipidemia Type V; hyperlipidemia Type VI; familial ligand-defectiveapo-B; familial hyperalphalipoproteinemia; hypobetalipoproteinemia,apolipoprotein B-100 deficiency; abetalipoproteinemia, Kornzweigsyndrome; and Tangier Disease, familial high-density lipoproteindeficiency.

In yet further preferred embodiments, the particulate delivery system ofthe present invention provides compositions and methods for thetreatment of mucopolysaccharide and glycolipid disorders, such as Type IH mucopolysaccharidosis (Hurler syndrome), Type I Smucopolysaccharidosis (Scheie syndrome), Type I H/Smucopolysaccharidosis (Hurler/Scheie syndrome), Type IImucopolysaccharidosis (Hunter's syndrome), Type IIImucopolysaccharidoses (Sanfilippo Type A, Sanfilippo Type B, SanfilippoType C, Sanfilippo Type D), Type IV mucopolysaccharidosis (Morquio'sType A, Morquio's Type B), Type VI mucopolysaccharidosis (Maroteaux-LamySyndrome) and Type VII mucopolysaccharidosis (Sly Syndrome).

In other preferred embodiments, the particulate delivery system of thepresent invention provides compositions and methods for the treatment ofdisorders of glycosphingolipid metabolism, such as GM1 gangliosidoses,including generalized GM1 Type II, juvenile form; generalized GM1 TypeIII, adult form; GM2 gangliosidosis, Sandhoff-Jatzkewitz disease; GM3gangliosidoses, Tay-Sachs disease, Tay-Sachs AB variant, Gaucherdisease, Niemann-Pick Disease, Types A, B, C1, C2 and D, Schindlerdisease, Fabry disease, lactosylceramidosis, Farber disease, Krabbedisease, multiple sulfatase deficiency, Austin disease, metachromicleukodystrophy, and sulfatide lipodosis.

In other preferred embodiments, the particulate delivery system of thepresent invention provides compositions and methods for the treatment ofoligosaccharidoses such as fucosidosis, mucolipodosis VI,sialolipidosis, alpha-mannosidosis, beta-mannosidosis, sialidoses TypesI and II, galactosialidosis, Goldberg syndrome andaspartylglucosaminuria.

In other preferred embodiments, the particulate delivery system of thepresent invention provides compositions and methods for the treatment ofdisorders of lysosomal enzyme transport such as mucolipidosis I,sialidosis; mucolipodosis II, I-cell disease; and mucolipodosis III,pseudo-Hurler polydystrophy.

In other preferred embodiments, the particulate delivery system of thepresent invention provides compositions and methods for the treatment ofdefects in amino acid and organic acid metabolism such asphenylketonuria; Type I tyrosinemia, tyrosinosis; Type II tyrosinemia,Richner-Hanhart syndrome; Type III tyrosinemia; alcaptonuria;homocystinuria; histidinemia; maple syrup urine disease (MSUD); MSUDType Ib, MSUD type II; methylmalonic aciduria; non-ketonichyperglycinemia Type I (NKHI) and hyperlysinemia.

In other preferred embodiments, the particulate delivery system of thepresent invention provides compositions and methods for the treatment ofurea cycle defects such as hyperammonemias; carbamoyl phosphatesynthetase I (CPS-I) deficiency; ornithine transcarbamylase (OTC)deficiency; N-acetylglutamate synthetase deficiency; argininosuccinicaciduria, argininosuccinate lyase deficiency; hyperargininemia, arginasedeficiency; citrullinemia, argininosuccinate synthetase deficiency andornithine aminotransferase deficiency. In other preferred embodiments,the particulate delivery system of the present invention providescompositions and methods for the treatment of defects in amino acidtransport such as cystinuria Type I; cystinuria Type III; Hartnupdisease and hyperammonemia-hyperornithinemia-homocitrullinuria (HHH)syndrome. In other preferred embodiments, the particulate deliverysystem of the present invention provides compositions and methods forthe treatment of porphyrias and bilirubinemias such as congenitalerythropoietic porphyria (CEP); erythropoietic protoporphyria (EPP); ALAdehydratase deficiency porphyria (ADP); acute intermittent porphyria(AIP); hereditary coproporphyria (HCP); variegate porphyria (VP);porphyria cutanea tarda (PCT); hepatoerythropoietic porphyria (HEP);Gilbert Syndrome; Crigler-Najjar Syndrome, Types I and I; Dubin-Johnsonsyndrome and Rotor syndrome.

In other preferred embodiments, the particulate delivery system of thepresent invention provides compositions and methods for the treatment oferrors in fatty acid metabolism such as very-long-chain acyl-CoAdehydrogenase deficiency (VLCAD); long-chain acyl-CoA dehydrogenasedeficiency (LCAD); medium-chain acyl-CoA dehydrogenase deficiency(MCAD); short-chain acyl-CoA dehydrogenase deficiency (SCAD; carnitinetranslocase deficiency; carnitine palmitoyltransferase I (CPT I)deficiency and carnitine palmitoylransferase II (CPT II) deficiency. Inother preferred embodiments, the particulate delivery system of thepresent invention provides compositions and methods for the treatment ofdefects in nucleotide metabolism such as Lesch-Nyhan syndrome; SevereCombined Immunodeficiency Disease (SCID), due to adenosine deaminase(ADA) deficiency; gout; renal lithiasis, due to adeninephosphoribosyltransferase (APRT) deficiency; xanthinuria, due toxanthine oxidase deficiency; orotic aciduria, Types I & I and ornithinetranscarbamoylase deficiency.

In other preferred embodiments, the particulate delivery system of thepresent invention provides compositions and methods for the treatment ofdisorders in metal metabolism and transport such as Wilson disease,Menkes disease, occipital horn syndrome and hemochromatosis. In otherpreferred embodiments, the particulate delivery system of the presentinvention provides compositions and methods for the treatment ofdisorders in peroxisomes such as Zellweger syndrome, X-linkedadreoleukodystrophy, neonatal adrenoleukodystophy (NALD), rhizomelicchondrodysplasia punctata (RCDP) and infantile Refsum's disease (IRD).In other preferred embodiments, the particulate delivery system of thepresent invention provides compositions and methods for the treatment ofdisorders associated with defective DNA repair such as ataxiatelangiectasia (AT), xeroderma pigmentosum (XP), Cockayne syndrome,Bloom syndrome and Fanconi anemia.

Routes of Administration

Routes of administration include but are not limited to oral; buccal,sublingual, pulmonary, transdermal, transmucosal, as well assubcutaneous, intraperitoneal, intravenous, and intramuscular injection.Preferred routes of administration are oral; buccal, sublingual,pulmonary and transmucosal.

The particulate delivery system of the present invention is administeredto a patient in a therapeutically effective amount. The particulatedelivery system can be administered alone or as part of apharmaceutically acceptable composition. In addition, a compound orcomposition can be administered all at once, as for example, by a bolusinjection, multiple times, such as by a series of tablets, or deliveredsubstantially uniformly over a period of time, as for example, using acontrolled release formulation. It is also noted that the dose of thecompound can be varied over time. The particulate delivery system can beadministered using an immediate release formulation, a controlledrelease formulation, or combinations thereof. The term “controlledrelease” includes sustained release, delayed release, and combinationsthereof.

A pharmaceutical composition of the invention can be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient that would be administeredto a patient or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the human treated and further depending upon theroute by which the composition is to be administered. By way of example,the composition can comprise between 0.1% and 100% (w/w) activeingredient. A unit dose of a pharmaceutical composition of the inventionwill generally comprise from about 100 milligrams to about 2 grams ofthe active ingredient, and preferably comprises from about 200milligrams to about 1.0 gram of the active ingredient.

In addition, a particulate delivery system of the present invention canbe administered alone, in combination with a particulate delivery systemwith a different payload, or with other pharmaceutically activecompounds. The other pharmaceutically active compounds can be selectedto treat the same condition as the particulate delivery system or adifferent condition.

If the patient is to receive or is receiving multiple pharmaceuticallyactive compounds, the compounds can be administered simultaneously orsequentially in any order. For example, in the case of tablets, theactive compounds may be found in one tablet or in separate tablets,which can be administered at once or sequentially in any order. Inaddition, it should be recognized that the compositions can be differentforms. For example, one or more compounds may be delivered via a tablet,while another is administered via injection or orally as a syrup.

Another aspect of the invention relates to a kit comprising apharmaceutical composition of the invention and instructional material.Instructional material includes a publication, a recording, a diagram,or any other medium of expression which is used to communicate theusefulness of the pharmaceutical composition of the invention for one ofthe purposes set forth herein in a human. The instructional material canalso, for example, describe an appropriate dose of the pharmaceuticalcomposition of the invention. The instructional material of the kit ofthe invention can, for example, be affixed to a container which containsa pharmaceutical composition of the invention or be shipped togetherwith a container which contains the pharmaceutical composition.Alternatively, the instructional material can be shipped separately fromthe container with the intention that the instructional material and thepharmaceutical composition be used cooperatively by the recipient.

The invention also includes a kit comprising a pharmaceuticalcomposition of the invention and a delivery device for delivering thecomposition to a human. By way of example, the delivery device can be asqueezable spray bottle, a metered-dose spray bottle, an aerosol spraydevice, an atomizer, a dry powder delivery device, a self-propellingsolvent/powder-dispensing device, a syringe, a needle, a tampon, or adosage-measuring container. The kit can further comprise aninstructional material as described herein.

For example, a kit may comprise two separate pharmaceutical compositionscomprising respectively a first composition comprising a particulatedelivery system and a pharmaceutically acceptable carrier; andcomposition comprising second pharmaceutically active compound and apharmaceutically acceptable carrier. The kit also comprises a containerfor the separate compositions, such as a divided bottle or a dividedfoil packet. Additional examples of containers include syringes, boxes,bags, and the like. Typically, a kit comprises directions for theadministration of the separate components. The kit form is particularlyadvantageous when the separate components are preferably administered indifferent dosage forms (e.g., oral and parenteral), are administered atdifferent dosage intervals, or when titration of the individualcomponents of the combination is desired by the prescribing physician.

An example of a kit is a blister pack. Blister packs are well known inthe packaging industry and are being widely used for the packaging ofpharmaceutical unit dosage forms (tablets, capsules, and the like).Blister packs generally consist of a sheet of relatively stiff materialcovered with a foil of a preferably transparent plastic material. Duringthe packaging process recesses are formed in the plastic foil. Therecesses have the size and shape of the tablets or capsules to bepacked. Next, the tablets or capsules are placed in the recesses and asheet of relatively stiff material is sealed against the plastic foil atthe face of the foil which is opposite from the direction in which therecesses were formed. As a result, the tablets or capsules are sealed inthe recesses between the plastic foil add the sheet. Preferably thestrength of the sheet is such that the tablets or capsules can beremoved from the blister pack by manually applying pressure on therecesses whereby an opening is formed in the sheet at the place of therecess. The tablet or capsule can then be removed via said opening.

It may be desirable to provide a memory aid on the kit, e.g., in theform of numbers next to the tablets or capsules whereby the numberscorrespond with the days of the regimen that the tablets or capsules sospecified should be ingested. Another example of such a memory aid is acalendar printed on the card, e.g., as follows “First Week, Monday,Tuesday, . . . etc. . . . Second Week, Monday, Tuesday,” etc. Othervariations of memory aids will be readily apparent. A “daily dose” canbe a single tablet or capsule or several pills or capsules to be takenon a given day. Also, a daily dose of a particulate delivery systemcomposition can consist of one tablet or capsule, while a daily dose ofthe second compound can consist of several tablets or capsules and viceversa. The memory aid should reflect this and assist in correctadministration.

In another embodiment of the present invention, a dispenser designed todispense the daily doses one at a time in the order of their intendeduse is provided. Preferably, the dispenser is equipped with a memoryaid, so as to further facilitate compliance with the dosage regimen. Anexample of such a memory aid is a mechanical counter, which indicatesthe number of daily doses that have been dispensed. Another example ofsuch a memory aid is a battery-powered micro-chip memory coupled with aliquid crystal readout, or audible reminder signal which, for example,reads out the date that the last daily dose has been taken and/orreminds one when the next dose is to be taken.

A particulate delivery system composition, optionally comprising otherpharmaceutically active compounds, can be administered to a patienteither orally, rectally, parenterally, (for example, intravenously,intramuscularly, or subcutaneously) intracisternally, intravaginally,intraperitoneally, intravesically, locally (for example, powders,ointments or drops), or as a buccal or nasal spray.

Parenteral administration of a pharmaceutical composition includes anyroute of administration characterized by physical breaching of a tissueof a human and administration of the pharmaceutical composition throughthe breach in the tissue. Parenteral administration thus includesadministration of a pharmaceutical composition by injection of thecomposition, by application of the composition through a surgicalincision, by application of the composition through a tissue-penetratingnon-surgical wound, and the like. In particular, parenteraladministration includes subcutaneous, intraperitoneal, intravenous,intraarterial, intramuscular, or intrasternal injection and intravenous,intraarterial, or kidney dialytic infusion techniques.

Compositions suitable for parenteral injection comprise the activeingredient combined with a pharmaceutically acceptable carrier such asphysiologically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions, or emulsions, or may comprise sterile powdersfor reconstitution into sterile injectable solutions or dispersions,Examples of suitable aqueous and nonaqueous carriers, diluents,solvents, or vehicles include water, isotonic saline, ethanol, polyols(propylene glycol, polyethylene glycol glycerol, and the like), suitablemixtures thereof, triglycerides, including vegetable oils such as oliveoil, or injectable organic esters such as ethyl oleate. Proper fluiditycan be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersions, and/or by the use of surfactants. Such formulations canbe prepared, packaged, or sold in a form suitable for bolusadministration or for continuous administration. Injectable formulationscan be prepared, packaged, or sold in unit dosage form, such as inampules, in multi-dose containers containing a preservative, or insingle-use devices for auto-injection or injection by a medicalpractitioner.

Formulations for parenteral administration include suspensions,solutions, emulsions in oily or aqueous vehicles, pastes, andimplantable sustained-release or biodegradable formulations. Suchformulations can further comprise one or more additional ingredientsincluding suspending, stabilizing, or dispersing agents. In oneembodiment of a formulation for parenteral administration, the activeingredient is provided in dry (i.e. powder or granular) form forreconstitution with a suitable vehicle (e.g. sterile pyrogen-free water)prior to parenteral administration of the reconstituted composition. Thepharmaceutical compositions can be prepared, packaged, or sold in theform of a sterile injectable aqueous or oily suspension or solution.This suspension or solution can be formulated according to the knownart, and can comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations can beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butanediol, for example. Other acceptable diluentsand solvents include Ringer's solution, isotonic sodium chloridesolution, and fixed oils such as synthetic mono- or di-glycerides. Otherparentally-administrable formulations which are useful include thosewhich comprise the active ingredient in microcrystalline form, in aliposomal preparation, or as a component of a biodegradable polymersystem. Compositions for sustained release or implantation can comprisepharmaceutically acceptable polymeric or hydrophobic materials such asan emulsion, an ion exchange resin, a sparingly soluble polymer, or asparingly soluble salt.

These compositions may also contain adjuvants such as preserving,wetting, emulsifying, and/or dispersing agents. Prevention ofmicroorganism contamination of the compositions can be accomplished bythe addition of various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, and the like. Itmay also be desirable to include isotonic agents, for example, sugars,sodium chloride, and the like. Prolonged absorption of injectablepharmaceutical compositions can be brought about by the use of agentscapable of delaying absorption, for example, aluminum monostearateand/or gelatin.

Dosage forms can include solid or injectable implants or depots. Inpreferred embodiments, the implant comprises an aliquot of theparticulate delivery system and a biodegradable polymer. In preferredembodiments, a suitable biodegradable polymer can be selected from thegroup consisting of a polyaspartate, polyglutamate, poly(L-lactide), apoly(D,L-lactide), a poly(lactide-co-glycolide), a poly(ε-caprolactone),a polyanhydride, a poly(beta-hydroxy butyrate), a poly(ortho ester) anda polyphosphazene.

Solid dosage forms for oral administration include capsules, tablets,powders, and granules. In such solid dosage forms, the particulatedelivery system is optionally admixed with at least one inert customaryexcipient (or carrier) such as sodium citrate or dicalcium phosphate or(a) fillers or extenders, as for example, starches, lactose, sucrose,mannitol, or silicic acid; (b) binders, as for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone,sucrose, or acacia; (c) humectants, as for example, glycerol; (d)disintegrating agents, as for example, agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain complex silicates, orsodium carbonate; (e) solution retarders, as for example, paraffin; (f)absorption accelerators, as for example, quaternary ammonium compounds;(g) wetting agents, as for example, cetyl alcohol or glycerolmonostearate; (h) adsorbents, as for example, kaolin or bentonite;and/or (i) lubricants, as for example, talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, or mixturesthereof. In the case of capsules and tablets, the dosage forms may alsocomprise buffering agents.

A tablet comprising the particulate delivery system can, for example, bemade by compressing or molding the active ingredient, optionally withone or more additional ingredients. Compressed tablets can be preparedby compressing, in a suitable device, the active ingredient in afree-flowing form such as a powder or granular preparation, optionallymixed with one or more of a binder, a lubricant, an excipient, a surfaceactive agent, and a dispersing agent. Molded tablets can be made bymolding, in a suitable device, a mixture of the active ingredient, apharmaceutically acceptable carrier, and at least sufficient liquid tomoisten the mixture. Pharmaceutically acceptable excipients used in themanufacture of tablets include inert diluents, granulating anddisintegrating agents, binding agents, and lubricating agents. Knowndispersing agents include potato starch and sodium starch glycolate.Known surface active agents include sodium lauryl sulfate. Knowndiluents include calcium carbonate, sodium carbonate, lactose,microcrystalline cellulose, calcium phosphate, calcium hydrogenphosphate, and sodium phosphate. Known granulating and disintegratingagents include corn starch and alginic acid. Known binding agentsinclude gelatin, acacia, pre-gelatinized maize starch,polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Knownlubricating agents include magnesium stearate, stearic acid, silica, andtalc.

Tablets can be non-coated or they can be coated using known methods toachieve delayed disintegration in the gastrointestinal tract of a human,thereby providing sustained release and absorption of the particulatedelivery system, e.g. in the region of the Peyer's patches in the smallintestine. By way of example, a material such as glyceryl monostearateor glyceryl distearate can be used to coat tablets. Further by way ofexample, tablets can be coated using methods described in U.S. Pat. Nos.4,256,108; 4,160,452; and 4,265,874 to form osmotically-controlledrelease tablets, Tablets can further comprise a sweetening agent, aflavoring agent, a coloring agent, a preservative, or some combinationof these in order to provide pharmaceutically elegant and palatablepreparation.

Solid dosage forms such as tablets, dragees, capsules, and granules canbe prepared with coatings or shells, such as enteric coatings and otherswell known in the art. They may also contain opacifying agents, and canalso be of such composition that they release the particulate deliverysystem in a delayed manner. Examples of embedding compositions that canbe used are polymeric substances and waxes. The active compounds canalso be in micro-encapsulated form, if appropriate, with one or more ofthe above-mentioned excipients.

Solid compositions of a similar type may also be used as fillers in softor hard filled gelatin capsules using such excipients as lactose or milksugar, as well as high molecular weight polyethylene glycols, and thelike. Hard capsules comprising the particulate delivery system can bemade using a physiologically degradable composition, such as gelatin.Such hard capsules comprise the particulate delivery system, and canfurther comprise additional ingredients including, for example, an inertsolid diluent such as calcium carbonate, calcium phosphate, or kaolin.Soft gelatin capsules comprising the particulate delivery system can bemade using a physiologically degradable composition, such as gelatin.Such soft capsules comprise the particulate delivery system, which canbe mixed with water or an oil medium such as peanut oil, liquidparaffin, or olive oil.

Oral compositions can be made, using known technology, whichspecifically release orally-administered agents in the small or largeintestines of a human patient. For example, formulations for delivery tothe gastrointestinal system, including the colon, include enteric coatedsystems, based, e.g., on methacrylate copolymers such aspoly(methacrylic acid, methyl methacrylate), which are only soluble atpH 6 and above, so that the polymer only begins to dissolve on entryinto the small intestine. The site where such polymer formulationsdisintegrate is dependent on the rate of intestinal transit and theamount of polymer present. For example, a relatively thick polymercoating is used for delivery to the proximal colon (Hardy et al., 1987Aliment. Pharmacol. Therap. 1:273-280). Polymers capable of providingsite-specific colonic delivery can also be used, wherein the polymerrelies on the bacterial flora of the large bowel to provide enzymaticdegradation of the polymer coat and hence release of the drug. Forexample, azopolymers (U.S. Pat. No. 4,663,308), glycosides (Friend etal., 1984, J. Med. Chem. 27:261-268) and a variety of naturallyavailable and modified polysaccharides (see PCT applicationPCT/GB89/00581) can be used in such formulations.

Pulsed release technology such as that described in U.S. Pat. No.4,777,049 can also be used to administer the particulate delivery systemto a specific location within the gastrointestinal tract. Such systemspermit delivery at a predetermined time and can be used to deliver theparticulate delivery system, optionally together with other additivesthat my alter the local microenvironment to promote stability anduptake, directly without relying on external conditions other than thepresence of water to provide in vive release.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirs. Inaddition to the active compounds, the liquid dosage form may containinert diluents commonly used in the art, such as water or othersolvents, isotonic saline, solubilizing agents and emulsifiers, as forexample, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethylformamide, oils, in particular, almond oil, arachis oil,coconut oil, cottonseed oil, groundnut oil, corn germ oil, olive oil,castor oil, sesame seed oil, MIGLYOL™, glycerol, fractionated vegetableoils, mineral oils such as liquid paraffin, tetrahydrofurfuryl alcohol,polyethylene glycols, fatty acid esters of sorbitan, or mixtures ofthese substances, and the like. Besides such inert diluents, thecomposition can also include adjuvants, such as wetting agents,emulsifying and suspending agents, demulcents, preservatives, buffers,salts, sweetening, flavoring, coloring and perfuming agents.Suspensions, in addition to the active compound, may contain suspendingagents, as for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol or sorbitan esters, microcrystalline cellulose, hydrogenatededible fats, sodium alginate, polyvinylpyrrdidone, gum tragacanth, gumacacia, agar-agar, and cellulose derivatives such as sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,aluminum metahydroxide, bentonite, or mixtures of these substances, andthe like. Liquid formulations of a pharmaceutical composition of theinvention that are suitable for oral administration can be prepared,packaged, and sold either in liquid form or in the form of a dry productintended for reconstitution with water or another suitable vehicle priorto use.

Known dispersing or wetting agents include naturally-occurringphosphatides such as lecithin, condensation products of an alkyleneoxide with a fatty acid, with a long chain aliphatic alcohol, with apartial ester derived from a fatty acid and a hexitol, or with a partialester derived from a fatty acid and a hexitol anhydride (e.g.polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylenesorbitol monooleate, and polyoxyethylene sorbitan monooleate,respectively). Known emulsifying agents include lecithin and acacia.Known preservatives include methyl, ethyl, orn-propyl-para-hydroxybenzoates, ascorbic acid, and sorbic acid. Knownsweetening agents include, for example, glycerol, propylene glycol,sorbitol, sucrose, and saccharin. Known thickening agents for oilysuspensions include, for example, beeswax, hard paraffin, and cetylalcohol.

In other embodiments, the pharmaceutical composition can be prepared asa nutraceutical, i.e., in the form of, or added to, a food (e.g., aprocessed item intended for direct consumption) or a foodstuff (e.g., anedible ingredient intended for incorporation into a food prior toingestion). Examples of suitable foods include candies such aslollipops, baked goods such as crackers, breads, cookies, and snackcakes, whole, pureed, or mashed fruits and vegetables, beverages, andprocessed meat products. Examples of suitable foodstuffs include milledgrains and sugars, spices and other seasonings, and syrups. Theparticulate delivery systems described herein are preferably not exposedto high cooking temperatures for extended periods of time, in order tominimize degradation of the compounds.

Compositions for rectal or vaginal administration can be prepared bymixing a particulate delivery system with suitable non-irritatingexcipients or carriers such as cocoa butter, polyethylene glycol or asuppository wax, which are solid at ordinary room temperature, butliquid at body temperature, and therefore, melt in the rectum or vaginalcavity and release the particulate delivery system. Such a compositioncan be in the form of, for example, a suppository, a retention enemapreparation, and a solution for rectal or colonic irrigation.Suppository formulations can further comprise various additionalingredients including antioxidants and preservatives. Retention enemapreparations or solutions for rectal or colonic irrigation can be madeby combining the active ingredient with a pharmaceutically acceptableliquid carrier. As is known in the art, enema preparations can beadministered using, and can be packaged within, a delivery deviceadapted to the rectal anatomy of a human. Enema preparations can furthercomprise various additional ingredients including antioxidants andpreservatives.

A pharmaceutical composition of the invention can be prepared, packaged,or sold in a formulation suitable for pulmonary administration via thebuccal cavity. Such compositions are conveniently in the form of drypowders for administration using a device comprising a dry powderreservoir to which a stream of propellant can be directed to dispersethe powder or using a self-propelling solvent/powder-dispensingcontainer such as a device comprising the particulate delivery systemsuspended in a low-boiling propellant in a sealed container. Dry powdercompositions may include a solid fine powder diluent such as sugar andare conveniently provided in a unit dose form. Low boiling propellantsgenerally include liquid propellants having a boiling point below 65degrees F. at atmospheric pressure. Generally the propellant canconstitute 50 to 99.9% (w/w) of the composition, and the activeingredient can constitute 0.1 to 20% (w/w) of the composition. Thepropellant can further comprise additional ingredients such as a liquidnon-ionic or solid anionic surfactant or a solid diluent (preferablyhaving a particle size of the same order as particles comprising theparticulate delivery system).

Pharmaceutical compositions of the invention formulated for pulmonarydelivery can also provide the active ingredient in the form of dropletsof a suspension. Such formulations can be prepared, packaged, or sold asaqueous or dilute alcoholic suspensions, optionally sterile, comprisingthe particulate delivery system, and can conveniently be administeredusing any nebulization or atomization device. Such formulations canfurther comprise one or more additional ingredients including aflavoring agent such as saccharin sodium, a volatile oil, a bufferingagent, a surface active agent, or a preservative such asmethylhydroxybenzoate.

The formulations described herein as being useful for pulmonary deliveryare also useful for intranasal delivery of a pharmaceutical compositionof the invention. Another formulation suitable for intranasaladministration is a coarse powder comprising the particulate deliverysystem. Such a formulation is administered in the manner in which snuffis taken i.e. by rapid inhalation through the nasal passage from acontainer of the powder held close to the nares.

A pharmaceutical composition of the invention can be prepared, packaged,or sold in a formulation suitable for buccal administration. Suchformulations can, for example, be in the form of tablets or lozengesmade using conventional methods, and can, for example, comprise 0.1 to20% (w/w) particulate delivery system, the balance comprising an orallydissolvable or degradable composition and, optionally, one or more ofthe additional ingredients described herein. Alternately, formulationssuitable for buccal administration can comprise a powder or anaerosolized or atomized solution or suspension comprising theparticulate delivery system.

Antibodies

As used herein, the term “antibody” (Ab) or “monoclonal antibody” (Mab)is meant to include intact molecules as well as antibody fragments (suchas, for example, Fab and F(ab′)2 fragments) which are capable ofspecifically binding to protein. Fab and F(ab′)2 fragments lack the Fcfragment of intact antibody, clear more rapidly from the circulation,and may have less non-specific tissue binding than an intact antibody.Thus, these fragments are preferred, as well as the products of a Fab orother immunoglobulin expression library. Moreover, antibodies of thepresent invention include chimeric, single chain, and humanizedantibodies.

Antibodies can be prepared using any number of techniques known in theart. Suitable techniques are discussed briefly below. The antibody maybe polyclonal or monoclonal. Polyclonal antibodies can have significantadvantages for initial development, including rapidity of production andspecificity for multiple epitopes, ensuring strong immunofluorescentstaining and antigen capture. Monoclonal antibodies are adaptable tolarge-scale production; preferred embodiments include at least onemonoclonal antibody specific for an epitope of the target antigen.Because polyclonal preparations cannot be readily reproduced forlarge-scale production, another embodiment uses a cocktail of at leastfour monoclonal antibodies.

A single chain Fv (“scFv” or “sFv”) polypeptide is a covalently linkedV_(H):V_(L) heterodimer which may be expressed from a nucleic acidincluding V_(H)- and V_(L)-encoding sequences either joined directly orjoined by a peptide-encoding linker. Huston, et al. Proc. Nat. Acad.Sci. USA, 85: 5879-5883 (1988). A number of structures for convertingthe naturally aggregated, but chemically separated, light and heavypolypeptide chains from an antibody V region into a scFv molecule whichfolds into a three dimensional structure substantially similar to thestructure of an antigen-binding site. See, e.g. U.S. Pat. Nos.6,512,097, 5,091,513 and 5,132,405 and 4,956,778.

In one class of embodiments, recombinant design methods can be used todevelop suitable chemical structures (linkers) for converting twonaturally associated, but chemically separate, heavy and lightpolypeptide chains from an antibody variable region into a sFv moleculewhich folds into a three-dimensional structure that is substantiallysimilar to native antibody structure. Design criteria includedetermination of the appropriate length to span the distance between theC-terminal of one chain and the N-terminal of the other, wherein thelinker is generally formed from small hydrophilic amino acid residuesthat do not tend to coil or form secondary structures. Such methods havebeen described in the art. See, e.g., U.S. Pat. Nos. 5,091,513 and5,132,405 to Huston et al.; and U.S. Pat. No. 4,946,778 to Ladner et al.

In this regard, the first general step of linker design involvesidentification of plausible sites to be linked. Appropriate linkagesites on each of the V_(H) and V_(L) polypeptide domains include thosewhich result in the minimum loss of residues from the polypeptidedomains, and which necessitate a linker comprising a minimum number ofresidues consistent with the need for molecule stability. A pair ofsites defines a “gap” to be linked. Linkers connecting the C-terminus ofone domain to the N-terminus of the next generally comprise hydrophilicamino acids which assume an unstructured configuration in physiologicalsolutions and preferably are free of residues having large side groupswhich might interfere with proper folding of the V_(H) and V_(L) chains.Thus, suitable linkers under the invention generally comprisepolypeptide chains of alternating sets of glycine and serine residues,and may include glutamic acid and lysine residues inserted to enhancesolubility. Nucleotide sequences encoding such linker moieties can bereadily provided using various oligonucleotide synthesis techniquesknown in the art.

Alternatively, a humanized antibody fragment may comprise the antigenbinding site of a murine monoclonal antibody and a variable regionfragment (lacking the antigen binding site) derived from a humanantibody. Procedures for the production of chimeric and furtherengineered monoclonal antibodies include those described in Riechmann etal. (Nature 332: 323, 1988), Liu et al. (PNAS 84: 3439, 1987), Larricket al. (Bio Technology 7: 934, 1989), and Winter and Harris (TIPS 14:139, May, 1993).

One method for producing a human antibody comprises immunizing anonhuman animal, such as a transgenic mouse, with a target antigen,whereby antibodies directed against the target antigen are generated insaid animal. Procedures have been developed for generating humanantibodies in non-human animals. The antibodies may be partially human,or preferably completely human. Non-human animals (such as transgenicmice) into which genetic material encoding one or more humanimmunoglobulin chains has been introduced may be employed. Suchtransgenic mice may be genetically altered in a variety of ways. Thegenetic manipulation may result in human immunoglobulin polypeptidechains replacing endogenous immunoglobulin chains in at least some(preferably virtually all) antibodies produced by the animal uponimmunization. Antibodies produced by immunizing transgenic animals witha target antigen are provided herein.

Mice in which one or more endogenous immunoglobulin genes areinactivated by various means have been prepared. Human immunoglobulingenes have been introduced into the mice to replace the inactivatedmouse genes. Antibodies produced in the animals incorporate humanimmunoglobulin polypeptide chains encoded by the human genetic materialintroduced into the animal. Examples of techniques for production anduse of such transgenic animals are described in U.S. Pat. Nos.5,814,318, 5,569,825, and 5,545,806, which are incorporated by referenceherein.

Monoclonal antibodies may be produced by conventional procedures, e.g.,by immortalizing spleen cells harvested from the transgenic animal aftercompletion of the immunization schedule. The spleen cells may be fusedwith myeloma cells to produce hybridomas, by conventional procedures.

A method for producing a hybridoma cell line comprises immunizing such atransgenic animal with a immunogen comprising at least seven contiguousamino acid residues of a target antigen; harvesting spleen cells fromthe immunized animal; fusing the harvested spleen cells to a myelomacell line, thereby generating hybridoma cells; identifying a hybridomacell line that produces a monoclonal antibody that binds a targetantigen. Such hybridoma cell lines, and monoclonal antibodies producedtherefrom, are encompassed by the present invention. Monoclonalantibodies secreted by the hybridoma cell line are purified byconventional techniques.

In another embodiment, antibody fragments are produced by selection froma nonimmune phage display antibody repertoire against one set ofantigens in the presence of a competing set of antigens (Stausbol-Grøn,B., et al., De novo identification of cell-type specificantibody-antigen pairs by phage display subtraction. Isolation of ahuman single chain antibody fragment against human keratin 14. Eur JBiochem 2001 May; 268(10):3099-107). This approach can be used toproduce phage antibodies directed against target antigens. The protocolin general is based on that described by Stausbol-Grøn, B., et al.,2001, Briefly, a nonimmunized semisynthetic phage display antibodyrepertoire is used. The repertoire is a single chain Fv (scFv) phagemidrepertoire constructed by recloning the heavy and light chain regionsfrom the lox library (Griffiths, A. D., et al. (1994) Isolation of highaffinity human antibodies directly from large synthetic repertoires.EMBO J. 13, 3245-3260). Escherichia coli TG1 (supE hsdD5 Δ(lac-proAB)thi F′ {traD36 proAB+ lacI^(q) lacZΔM15]) is an amber suppressor strain(supE) and is used for propagation of phage particles. E. coli HB2151(ara A(lac-proAB) thi F {proAB+ lacI^(q) lacZΔM15]) is a nonsuppressorstrain and is used for expression of soluble scFv. In anotherembodiment, a human single-chain Fv (scFv) library can be amplified andrescued, as described (Gao, at al., Making chemistry selectable bylinking it to infectivity, Proc. Natl. Acad. Sci. USA, Vol. 94, pp.11777-11782, October 1997). The library is panned against targetantigens suspended in PBS (10 mM phosphate, 150 mM NaCl, pH 7.4) and thepositive scFv-phage are selected by enzyme-linked immunosorbent assay(EUSA).

In other preferred embodiments, an antibody is supplied by providing anexpression vector encoding a recombinant antibody, preferably a singlechain Fv antibody.

Example 1

FIG. 1 is a schematic diagram 100 of a transverse section of a yeastcell wall, showing, from outside to inside, an outer fibrillar layer110, an outer mannoprotein layer 120, a beta glucan layer 130, a betaglucan layer-chitin layer 140, an inner mannoprotein layer 150, theplasma membrane 160 and the cytoplasm 170.

Preparation of WGP Particles

Whole Glucan Particles (WGP, Lot W0282) were previously obtained fromAlpha-Beta Technology. In general, whole glucan particles are preparedfrom yeast cells by the extraction and purification of thealkali-insoluble glucan fraction from the yeast cell walls. The yeastcells are treated with an aqueous hydroxide solution without disruptingthe yeast cell walls, which digests the protein and intracellularportion of the cell, leaving the glucan wall component devoid ofsignificant protein contamination, and having substantially theunaltered cell wall structure of β(1-6) and β(1-3) linked glucans. Yeastcells (S. cerevisae strain R4) were grown to midlog phase in minimalmedia under fed batch fermentation conditions. Cells (˜90 g dry cellweight/L) were harvested by batch centrifugation at 2000 rpm for 10minutes. The cells were then washed once in distilled water and thenresuspended in 1 liter of 1M NaOH and heated to 90 degrees Celsius. Thecell suspension was stirred vigorously for 1 hour at this temperature.The insoluble material, containing the cell walls, was recovered bycentrifuging at 2000 rpm for 10 minutes. This material was thensuspended in 1 liter, 1M NaOH and heated again to 90 degrees Celsius.The suspension was stirred vigorously for 1 hour at this temperature.The suspension was then allowed to cool to room temperature and theextraction was continued for a further 16 hours. The insoluble residuewas recovered by centrifugation at 2000 rpm for 10 minutes. Thismaterial was finally extracted in 1 liter, water brought to pH 4.5 withHCl, at 75 degrees Celsius for 1 hour. The insoluble residue wasrecovered by centrifugation and washed three times with 200 milliliterswater, four times with 200 milliliters isopropanol and twice with 200milliliters acetone. The resulting slurry was placed in glass trays anddried at 55 degrees Celsius under reduced pressure to produce 7.7 g of afine white powder.

A more detailed description of whole glucan particles and a process ofpreparing them can be found in U.S. Pats. Nos. 4,810,646; 4,992,540;5,028,703; 5,607,677 and 5,741,495, the teachings of which areincorporated herein by reference. For example, U.S. Pat. No. 5,028,703discloses that yeast WGP particles can be produced from yeast cells infermentation culture. The cells were harvested by batch centrifugationat 8000 rpm for 20 minutes in a Sorval RC2-B centrifuge. The cells werethen washed twice in distilled water in order to prepare them for theextraction of the whole glucan. The first step involved resuspending thecell mass in 1 liter 4% w/v NaOH and heating to 100 degrees Celsius. Thecell suspension was stirred vigorously for 1 hour at this temperature.The insoluble material containing the cell walls was recovered bycentrifuging at 2000 rpm for 15 minutes. This material was thensuspended in 2 liters, 3% w/v NaOH and heated to 75 degrees Celsius. Thesuspension was stirred vigorously for 3 hours at this temperature. Thesuspension was then allowed to cool to room temperature and theextraction was continued for a further 16 hours. The insoluble residuewas recovered by centrifugation at 2000 rpm for 15 minutes. Thismaterial was finally extracted in 2 liters, 3% w/v NaOH brought to pH4.5 with HCl, at 75 degrees Celsius for 1 hour. The insoluble residuewas recovered by centrifugation and washed three times with 200milliliters water, once with 200 milliliters dehydrated ethanol andtwice with 200 milliliters dehydrated ethyl ether. The resulting slurrywas placed on petri plates and dried.

Preparation of YGMP Particles

S. cerevisiae (100 g Fleishmans Bakers yeast) was suspended in 1 liter1M NaOH and heated to 55 degrees Celsius. The cell suspension was mixedfor 1 hour at this temperature. The insoluble material containing thecell walls was recovered by centrifuging at 2000 rpm for 10 minutes.This material was then suspended in 1 liter of water and brought to pH4-5 with HCl, and incubated at 55 degrees Celsius for 1 hour. Theinsoluble residue was recovered by centrifugation and washed once with1000 milliliters water, four times with 200 milliliters dehydratedisopropanol and twice with 200 milliliters acetone. The resulting slurrywas placed in a glass tray and dried at room temperature to produce 12.4g of a fine, slightly off-white, powder.

Preparation of YGMP Particles

S. cerevisiae (75 g SAF-Mannan) was suspended in 1 liter water andadjusted to pH 12-12.5 with 1M NaOH and heated to 55 degrees Celsius.The cell suspension was mixed for 1 hour at this temperature. Theinsoluble material containing the cell walls was recovered bycentrifuging at 2000 rpm for 10 minutes. This material was thensuspended in 1 liter of water and brought to pH 4-5 with HCl, andincubated at 55 degrees Celsius for 1 hour. The insoluble residue wasrecovered by centrifugation and washed once with 1000 milliliters water,four times with 200 milliliters dehydrated isopropanol and twice with200 milliliters acetone. The resulting slurry was placed in a glass trayand dried at room temperature to produce 15.6 g of a fine slightlyoff-white powder.

Preparation of YCP Particles

Yeast cells (Rhodotorula sp.) derived from cultures obtained from theAmerican Type Culture Collection (ATCC, Manassas, Va.) were aerobicallygrown to stationary phase in YPD at 30 degrees Celsius. Rhodotorula sp.cultures available from ATCC include Nos. 886, 917, 9336, 18101, 20254,20837 and 28983. Cells (1L) were harvested by batch centrifugation at2000 rpm for 10 minutes. The cells were then washed once in distilledwater and then resuspended in water brought to pH 4.5 with HCl, at 75degrees Celsius for 1 hour. The insoluble material containing the cellwalls was recovered by centrifuging at 2000 rpm for 10 minutes. Thismaterial was then suspended in 1 liter, 1M NaOH and heated to 90 degreesCelsius for 1 hour. The suspension was then allowed to cool to roomtemperature and the extraction was continued for a further 16 hours. Theinsoluble residue was recovered by centrifugation at 2000 rpm for 15minutes and washed twice with 1000 milliliters water, four times with200 milliliters isopropanol and twice with 200 milliliters acetone. Theresulting slurry was placed in glass trays and dried at room temperatureto produce 2.7 g of a fine light brown powder.

FIG. 2A is a diagram of the structure of a yeast cell wall particle;FIG. 2B is a fluorescence photomicrograph showing concanavalin-A-FITC(con-A-fluorescein isothiocyanate, Sigma Chemical, St. Louis, Mo.)staining of the mannan component of the yeast cell wall particles; FIG.2C is a diagram of the structure of a YGMP beta glucan-mannan particle,FIG. 2D is a fluorescence photomicrograph showing punctuate con-A-FITCstaining of a YGMP beta glucan-mannan particle; FIG. 2E is a diagram ofthe structure of a YGP beta glucan particle and FIG. 2F is afluorescence micrograph showing the absence of con-A-FITC staining of aYGP beta glucan particle.

Concanavalin-A is a lectin that binds selectively to mannose.Concanavalin-A-FITC binding was evaluated by fluorescence microscopy inorder to observe the amount and distribution pattern of mannan on thesurface of various yeast cell wall preparations. Suspensions of Baker'syeast (Fleishmans Bakers yeast), YGMP and YGP in PBS+1 mM MgCl₂+1 mMCaCl₂ were prepared at a density of 1×10⁸ particles/ml. Con-A-FITC stockwas 1 mg/ml concanavalin-A-FITC in PBS+1 mM MgCl₂+1 mM CaCl₂. Labelingmixtures were prepared in microcentrifuge tubes consisting of:

100 μl PBS+1 mM MgCl₂+1 mM CaCl₂

2.5 μl yeast cell wall particle suspension

2.5 μl con-A-FITC stock solution.

The microcentrifuge tubes containing the labeling mixtures wereincubated in the dark at room temperature for one hour. Yeast cell wallparticles were collected by centrifugation (10,000 rpm for 10 minutes)followed by washing the pellet with 100 μl PBS three times. The washedyeast cell wall particles were resuspended in 100 μl PBS and transferredto a 96 well plate for examination with a fluorescence microscope.Photographs of exemplary fields are shown in FIGS. 2B, 2D and 2F.

Table 1 summarizes the results of analyses of the chemical compositionof WGP particles, YGP particles, YGMP particles and YCP particles thatwere prepared as described above. Note that YGP particles and YGMPparticles have lower beta-glucan higher protein compared to the priorart WGP particles. YGMP particles have a substantially higher mannancontent compared to the other particle types. YCP particles have asubstantially higher chitin+chitosan content compared to the otherparticle types.

TABLE 1 Chemical Composition of Yeast Cell Wall Materials WGP YGMP YGPS. S. S. YCP Analyte Method cerevisiae cerevisiae cerevisiae RhodotoruiaMacromolecular Composition* Protein Kjeldal <1 4.5 4.9 — Fat Base <1 1.61.4 — hydrolysis, Soxhlet extraction Ash Combustion 1.2 1.9 1.6 —Carbohydrate Composition** Beta-Glucan Enzymatic 90.3 41.9 77 6.5Hydrolysis Chitin + chitosan Monosac 2.1 2.3 2.4 68 (as glocosamine,n-acetyl Analysis- glucosamine) Dionex Mannan Monosac <1 36.9 0.47 1.3(as mannose) Analysis- Dionex Other Glucans Monosac 6.2 10.9 11.2 0.2(as non beta 1,3-glucose Analysis- and other unmeasured Dionex sugars)*Results are reported % w/w of dry analyzed materials **Results arereported % w/w carbohydrate WGP—Whole Glucan Particle—Prior ArtTechnology YGMP—Yeast Glucan-Mannan Particle YGP—Yeast Glucan ParticleYCP—Yeast Chitin Particle

Example 2 Hydrodynamic Volume of Yeast Cell Wall Particles

The hydrodynamic volume of yeast cell wall particles was determined as ameasure of the payload capacity of the particles. A 1 g aliquot of yeastcell wall particles was weighed in a tared 15 ml centrifuge tube todetermine the weight of the dry particles. Water (12.5 mil) was added tothe tube, and the tube was vortexed to mix the suspension of yeast cellwall particles. The particles were allowed to swell and absorb water for30 minutes. The particle suspension was centrifuged at 2000 rpm for 10minutes. The water was removed, the tube was weighed, and the weight ofwater absorbed was calculated. The hydrodynamic volume was calculated asthe ratio of the weight of the water absorbed to the weight of the dryparticles. Table 2 presents the results for two preparations of theprior art WGP and the YGP and YGMP of the present invention.

TABLE 2 Hydrodynamic Volume of Exemplary Yeast Cell Wall PreparationsYeast Cell Wall Hydrodynamic Volume Particle (g water/g particles) WGPPrep 1 9.7 WGP Prep 2 6.9 YGP 8.3 YGMP 6.7

The lower hydrodynamic volume of WGP Prep 2 may be due to an increasednumber of fragmented particles in this preparation. With respect to theother particles, the “purer” YGP had a higher hydrodynamic volume thanthe YGMP.

In general, the payload volume was limited to <66% hydrodynamic volumeto ensure quantitative absorption of the payload by the yeast cell wallparticles. By this rule, ≦5.5 μl payload would be loaded per mg YGPparticles and ≦4.4 μl payload would be loaded per mg YGMP particles.

Example 3 Oral Bioavailability of YGP and YGMP

Fluorescently labeled yeast glucan particles (YGP-F) and fluorescentlylabeled yeast glucan-mannan particles (YGMP-F) were prepared for anuptake study. Starting materials were: 5 ml YGP (5 mg/ml in 0.1M boratebuffer, pH 8), 5 ml YGMP (5 mg/ml in 0.1M borate buffer, pH8),dichlorotriazinyl aminofluorescein (DTAF), 20 mg/ml in DMSO, freshlyprepared and 0.1M borate buffer, pH 8.

Labeling reactions were carried out at a 25 mg scale. Aliquots of 25 mgparticles were suspended in 5 ml 0.1M borate buffer, pH 8 and sonicatedto reduce clumps of particles to single particles. The particles werecentrifuged and resuspended in 5 ml 0.1M borate buffer, pH 8. DTAF (0.5ml 20 mg/ml) was added to the resuspended particles and incubated 2 daysat 37 degrees Celsius. At the end of the incubation, 5 ml 1 M Trisbuffer, pH 8.3, was added and the mixture was incubated 30 minutes toquench DTAF. The incubated particles were centrifuged and washed in PBSuntil the supernatants were no longer fluorescent. The washed particleswere resuspended in PBS at 5 mg/ml. The number of particles in a 1:100dilution of an aliquot was counted. Results: intensely fluorescent yeastcell wall particles were produced, at concentrations of 1.8×10⁹particles per ml YGP-F and 2.1×10⁹ particles per ml YGMP-F.

The influence of the surface carbohydrate composition on the oralbioavailability of yeast glucan particles was studied to determine ifthe phagocytic particle uptake of a payload could be targeted via themannose receptor as well as by the CR3/dectin-1 beta glucan receptors.The ability to target either or both receptors can expand the targetpopulation of cells beyond macrophages and dendritic cells.

The treatment groups are summarized in Table 3, below. Startingmaterials included: FITC-labeled yeast glucan particles (YGP-F),FITC-labeled yeast glucan-mannan particles (YGMP-F), a group of sevenC57Black mice and a group of seven C57/B16 mice. Doses of YGP-F (1mg/ml) and YGMP-F (3.7 mg/ml) were prepared to deliver equivalent numberof particles in 0.1 ml PBS and administered by oral gavage to one mousefrom each group daily for five days. The same dose was administered bysubcutaneous injection of 0.1 ml to one mouse from each group daily forfive days. On day four the cages were changed and fresh bedding wasprovided. Fecal pellets were collected on day 5 from each group into 15ml conical tubes and frozen for processing later. The fecal pellets wereprocessed by adding 5 ml water and holding at 4 degrees Celsius for 2hours. The hydrated fecal pellets were homogenized using a Polytronhomogenizer. Dilutions of homogenized feces were placed in a 96-wellmicrotiter plate and microscopically examined under fluorescent andtransmitted white light conditions for the presence of fluorescentparticles. Aliquots having fluorescent particles were further dilutedand the number of fluorescent particles/ml was counted with ahematocytometer.

Mice were sacrificed on day 7, and the spleen was removed from eachanimal and placed into separate tubes containing PBS on ice. The spleenswere macerated with scissors and pressed through 70 micron screens toproduce single cell suspensions. Aliquots of the single cell suspensionswere retained and fixed in 1% formalin in PBS for quantifying thefraction of cells labeled with fluorescent particles using FACS. Cellsuspensions are stained using a phycoerythrin (PE) labeled-antibodyagainst macrophage marker, preferably murine Emr-1 (F4/80), which stainssplenic red pulp macrophages, Kupffer cells, microglia and Langerhanscells.

Cell suspensions were plated at a density of 10⁷ cells per 60 mm petridish in DMEM containing 10% fetal calf serum (JRH Scientific),penicillin-streptomycin and glutamine (Gibco) and incubated for 24 hoursat 37 degrees Celsius under 5% CO₂ to allow for attachment. After theincubation, any unattached lymphocytes were washed away. The attachedsplenic macrophage cells were typsinized, fixed and scored for thefraction of adherent cells having fluorescent particles using afluorescence microscope.

The administration of the fluorescent particles was well tolerated.Analysis of adherent splenic macrophages demonstrated the presence offluorescent yeast cell wall particles in all fluorescent particletreated animals. These results demonstrate that both YGP-F and YGMP-Fare orally bioavailable and can be systemically distributed bymacrophages. The analysis of feces demonstrated the presence offluorescent particles, indicating that oral absorption was incomplete atthe dosage levels used. C57/B16 mice were able to absorb YGP-F andYGMP-F administered orally. The number of fluorescent particles in feceswas quantified as an estimate of uptake efficiency.

TABLE 3 Presence of Fluorescent Particles # # Splenic Route TreatmentDose mg/ml part./ml part./dose Macrophages Feces Control PBS — — —control SQ YGP-F 100 μg 1 1 × 10⁹ 1 × 10⁸ + − Oral YGP-F 100 μg 1 1 ×10⁹ 1 × 10⁸ + + Oral YGP-F  33 μg 0.33 3.3 × 10⁸   3.3 × 10⁷   + + SQYGPM-F   370 μg 3.7 1 × 10⁹ 1 × 10⁸ + − Oral YGPM-F   370 μg 3.7 1 × 10⁹1 × 10⁸ + + Oral YGPM-F   110 μg 1.1 1 × 10⁸ 3.3 × 10⁷   + + Untreated —— — — — − − Control

Example 4 Preparation of Chitosan Loaded YGP Particles

YGP particles were prepared with a cationic trapping polymer, chitosan.1% w/v chitosan solutions were prepared in 0.1M acetic acid using eitherHigh Molecular Weight (HMW) chitosan (˜70,000 Mw, Sigma Chemical St.Louis, Mo.) or Low Molecular Weight (HMW) chitosan (˜10,000 Mw, SigmaChemical St. Louis, Mo.). Both 1% w/v HMW and LMW chitosan solutionswere prepared in 0.1M acetic acid. Four ml HMW or LMW chitosan solutionwas added to 2 g YGP in a 50 ml conical centrifuge tube and mixed untila smooth paste was formed. The mixture was incubated for 1 hour at roomtemperature to allow the liquid to be absorbed. NaOH (40 ml, 0.1M) wasadded to each tube, which was vortexed immediately to precipitate thechitosan inside the YGP. The YGP:chitosan suspension was passed throughan 18 gauge needle to produce a fine suspension of YGP:chitosanparticles. The YGP:chitosan particles were collected by centrifugation(2,000 rpm for 10 minutes) followed by washing the pellet with deionizedwater until the pH of the supernatant was 7-8. The YGP:chitosanparticles were then washed four times with two pellet volumes ofisopropanol and then washed twice with two pellet volumes of acetone.The YGP:chitosan particles were then dried at room temperature in ahood. The procedure yielded 1.2 g YGP:LMW chitosan particles and 1.4 gYGP:HMW chitosan particles.

Example 5 Preparation of CytoPure™ Loaded YGP Particles

YGP particles were prepared with a biodegradable cationic trappingpolymer, CytoPure™, a proprietary, commercially available, water-solublecationic polymer transfection reagent (Qbiogene, Inc., CA). Twenty μlCytoPure™ was diluted in 0.5 ml deionized water and added to 0.5 g YGPin a 50 ml conical centrifuge tube and mixed until a smooth paste wasformed. The mixture was incubated for 15 minutes at 4 degrees Celsius toallow the liquid to be absorbed. Twenty-five ml ethanol was added toeach tube, which was vortexed immediately to precipitate the CytoPure™inside the YGP. The YGP:CytoPure™ suspension was sonicated to produce afine suspension of YGP:CytoPure™ particles. The YGP:CytoPure™ particleswere collected by centrifugation (2,000 rpm for 10 minutes) followed bywashing the pellet four times with two pellet volumes of isopropanol andthen washed twice with two pellet volumes of acetone. The YGP:CytoPure™particles were then dried at room temperature in a hood. The procedureyielded 0.45 g YGP:CytoPure™ particles.

Example 6 Preparation of Polyethylenimine Loaded YGP Particles

YGP particles were prepared with polyethylenimine (PEI) as a cationictrapping polymer. A 0.5 ml aliquot of a 2% w/v PEI (˜50,000 Mw, SigmaChemical Co., St. Louis, Mo.) solution in water was added to 0.5 g YGPin a 50 ml conical centrifuge tube and mixed until a smooth paste wasformed. The mixture was incubated for one hour at room temperature toallow the liquid to be absorbed. Twenty-five ml ethanol was added toeach tube, which was vortexed immediately to precipitate the PEI insidethe YGP. The YGP:PEI suspension was passed through an 18 gauge needle toproduce a fine suspension of YGP:PEI particles. The YGP:PEI particleswere collected by centrifugation (2,000 rpm for 10 minutes) followed bywashing the pellet four times with two pellet volumes of isopropanol andthen washed twice with two pellet volumes of acetone. The YGP:PEIparticles were then dried at room temperature in a hood. The procedureyielded 0.48 g YGP:PEI particles.

Example 7 Preparation of Alginate Loaded YGP Particles

YGP particles were prepared with alginate (F200 or F200L, Multi-KemCorp., Ridgefield, N.J.) as an anionic trapping polymer. A 2 ml aliquotof a 1% w/v alginate solution in water was added to 1 g YGP in a 50 mlconical centrifuge tube and mixed to form a smooth paste. The mixturewas incubated for one hour at room temperature to allow the liquid to beabsorbed. The mixture was diluted with 40 ml of a 1% w/v calciumchloride aqueous solution. The YGP:alginate suspension was passedthrough an 18 gauge needle to produce a fine suspension of YGP:alginateparticles. The YGP:alginate particles were collected by centrifugation(2,000 rpm for 10 minutes. The YGP:alginate particles were washed fourtimes with two pellet volumes of isopropanol and then washed twice withtwo pellet volumes of acetone. The YGP:alginate particles were thendried at room temperature in a hood. The procedure yielded 0.95 gYGP:F200 alginate particles and 0.86 g YGP:F200L alginate particles.

Example 8 Preparation of Poly-L-lysine Loaded YGP and YGMP Particles

YGP and YGMP particles were prepared with Poly-L-lysine (PLL) as atrapping polymer. A 4 ml aliquot of a 1% w/v PLL (Sigma Chemical Co.,St. Louis, Mo.) solution in water was added to 1 g YGP or YGMP in a 50ml conical centrifuge tube. The mixture was incubated for 30 minutes at55 degrees Celsius to allow the liquid to be absorbed. Ten ml ethanolwas added to each tube, which was homogenized (Polytron homogenizer) toproduce a fine suspension of YGP:PLL or YGMP:PLL particles. The YGP:PLLor YGMP:PLL particles were collected by centrifugation (2,000 rpm for 10minutes. The YGP:PLL or YGMP:PLL were washed four times with two pelletvolumes of isopropanol and then washed twice with two pellet volumes ofacetone. The YGP:PLL or YGMP:PLL particles were then dried at roomtemperature in a hood. The procedure yielded 1.3 g YGP:PLL particles and1.1 g YGMP:PLL particles. Microscopic evaluation showed no free PLLaggregates, only YGP:PLL or YGMP:PLL particles.

Example 9 Preparation of Xanthan Loaded YGP and YGMP Particles

YGP and YGMP particles were prepared with xanthan as an anionic trappingpolymer. A 4 ml aliquot of a 1% w/v xanthan solution in water was heatedto 55 degrees Celsius to reduce viscosity and added to 1 g YGP or YGMPin a 50 ml conical centrifuge tube. The mixture was incubated for 30minutes at 55 degrees Celsius. Ten ml ethanol was added to each tube,which was homogenized (Polytron homogenizer) to produce a finesuspension of YGP:xanthan or YGMP:xanthan particles. The YGP:xanthan orYGMP:xanthan particles were collected by centrifugation (2,000 rpm for10 minutes). The YGP:xanthan or YGMP:xanthan particles were washed fourtimes with two pellet volumes of isopropanol and then washed twice withtwo pellet volumes of acetone. The YGP:xanthan or YGMP:xanthan particleswere then dried at room temperature in a hood. The procedure yielded 1.2g YGP:xanthan particles and 1.1 g YGMP:xanthan particles. Microscopicevaluation showed no free xanthan aggregates, only YGP:xanthan orYGMP:xanthan particles.

Example 10 Evaluation of Ability of YGP:Chitosan and YGP:Alginate ToBind Charged Dyes

YGP:Chitosan and YGP:Alginate particles were prepared as described inExamples 7 & 9 above. 0.1% w/v aqueous solutions of trypan blue(Benzamine blue; CI 23850), an anionic dye and xylene cyanol (acid blue,a cationic dye) were prepared. A 50 μl aliquot of a 0.1% w/v aqueous dyesolution was added to 10 mg YGP, YGP:Chitosan or YGP:Alginate inmicrocentrifuge tubes and the mixture was incubated for 1 hour at roomtemperature. The pellets were washed with deionized water until thesupernatant solutions were no longer colored. The color of the pelletwas evaluated; the results are presented in Table 4, below.

TABLE 4 Pallet Color YGP Formulation Trypan blue Xylene cyanol YGP TanTan YGP:Chitosan Blue Tan YGP:Alginate Tan Green

Electrostatic interactions between insoluble trapping polymers insideYGP were capable of binding to oppositely charged low molecular weightmodel dye payloads.

Example 11 Use of YGP:Agarose to Trap Molecules by Physical Entrapment

YGP:Agarose was prepared to evaluate physical entrapment as a means totrap a payload in YGP. A 2% w/v solution of agarose (Sigma Chemical Co.,St. Louis, Mo.) was prepared in TE, and cooled to 50 degrees Celsius. A1 mg/ml stock solution of salmon sperm DNA in TE was diluted to 0.5mg/ml DNA in TE or in 1% agarose at 50 degrees Celsius. A 500 mg aliquotof YGP was mixed with 500 al of DNA in TE or 500 μl of DNA in agarose at50 degrees Celsius and the mixture was incubated 1 hour at 50 degreesCelsius. The mixture was then cooled for 1 hour in a refrigerator tosolidify the agarose. After 1 hour, 10 mls of TE was added and themixture was incubated overnight in refrigerator, The mixture was thencentrifuged, and DNA in the supernatant was measured by absorption at260 nm. About >80% of the applied DNA was retained by YGP:Agarosecompared to <1% retained by the YGP:TE control. These results indicatethat agarose effectively traps DNA inside YGP by physical entrapment.

Example 12 Use of YGP:Polyacrylamide to Trap Molecules by PhysicalEntrapment

YGP:Polyacrylamide was prepared to evaluate physical entrapment as ameans to trap a payload in YGP. A 1 mg/mil stock solution of salmonsperm DNA in TE was diluted to 0.5 mg/ml DNA in TE or in 30%polyacrylamide/bis(Sigma Chemical Co., St. Louis, Mo.). TEMED(N,N,N′,N′-Tetramethylethylenediamine) was added to each DNA mixture (1μl TEMED to 5 mls of DNA solution), and a 2 ml aliquot of each solutionwas added to 1 g YGP. The result was mixed to form a uniform suspensionand incubated 3 hours at room temperature. After the 3 hour incubation,10 ml of TE was added and the mixture was incubated overnight in arefrigerator. The mixture was then centrifuged, and DNA in thesupernatant was measured by absorption at 260 nm. About >95% of theapplied DNA was retained by YGP:Polyacrylamide compared to <1% retainedby the YGP:TE control. These results indicate that polyacrylamide is aneffective trapping polymer to use to trap DNA inside YGP by physicalentrapment.

Example 13 Loading YGP With A Small Molecule, Tetracycline

The antibiotic tetracycline (tet) was loaded into YGP using the relativeinsolubility of the tetracycline-calcium salt. Yeast cell wall particlesused were YGP, YGP:F200 alginate and YGP: F200L alginate prepared asdescribed above. Stock solutions were 1 M CaCl₂ and 100 mg/mltetracycline HCl (Sigma Chemical Co., St. Louis, Mo.). The loadingmixtures were set up as summarized in Table 5, below.

TABLE 5 Loading Release A355* % tet % tet A355 YGP (1 mg) Tet (μl) Water(μl) 1 M CaCl₂ (μl) super bound w/w PBS 0.1 M HCl — — — 200 0 — — — — —4 200 — 0.538 — — — — — 4 — 200 0.542 — — — — YGP — — 200 0.01 — — — —YGP 4 200 — 0.56 0 — — — YGP 4 — 200 0.524 <1 — — — YGP-F200 4 200 —0.405 24.8 9.9 3.6 4.9 alginate YGP-F200L 4 200 — 0.375 30.3 12.1 5.912.2 alginate * 1/100 dilution

The mixtures were incubated for 30 minutes at room temperature and thendeionized water or 1 M CaCl₂ was added as indicated. After 60 minutes atroom temperature, the mixtures were sonicated and were incubated for atleast an additional 30 minutes at room temperature. The mixtures werethen centrifuged (2,000 rpm for 10 minutes) and the presence oftetracycline was indicated by the yellow color of the pellet and that ofthe initial supernatant. The amount of tetracycline loading into theyeast cell wall particles was calculated from the loss of absorption at355 nm, the peak of the tetracycline absorption spectrum. A dilution of4 μl of the 100 mg/ml tetracycline HCl stock solution in 200 μldeionized water had an absorbance at 355 nm of 0.538 compared to adeionized water blank. Release of tetracycline from the loaded yeastcell wall particles into PBS or 0.1M HCl was also measuredspectrophotometrically.

The results are summarized in Table 5, above. In general, while YGP:F200alginate and YGP:F200L alginate pellets were yellow after washing, YGPpellets were not yellow, indicating little, if any, tetracycline loadingeither a the hydrochloride or the calcium salt in the absence of atrapping polymer. In contrast, tetracycline was effectively loaded andtrapped in YGP:F200 alginate and YGP:F200L alginate formulations, withabout 25-30% of the applied tetracycline load absorbed as the calciumalginate salt. Trapped tetracycline was released from YGP:F200 alginateand YGP:F200L alginate into 0.1M HCl. The trapped tetracycline waspartially retained in YGP:F200 alginate and YGP:F200L alginate in PBSfor 1 hour at 37 degrees Celsius, about 26.5-51.6% of 0.1M HClextractable.

In summary, tetracycline was readily trapped as a calcium alginate saltcomplex in a YGP-alginate-calcium composition, but was not effectivelyloaded and retained within YGP alone. The tetracycline trapped as acalcium alginate complex in YGP:F200 alginate and YGP:F200L alginate wasslowly released in PBS at 37 degrees Celsius and substantially releasedunder acid conditions.

Example 14 Efficacy of Tet and YGP:Tet In Increasing in vitroMicrobiocidal Killing of J774 Macrophages

YGP:alginate-tet was prepared as described in Example 13, above. Thenumbers of particles of YGP and YGP: alginate-tet per ml in the stocksolutions were 9×10⁷/ml and 6×10⁸/ml, respectively.

TABLE 6 S. aureus Killing By J774 Murine Macrophages Loaded With YGPParticles Fold YGP J774 YGP/tet S. aureus Increased Tube 5 × 10⁵/mlDMEM + C 5 × 10⁷/ml μl Particles/ml Killed Killing a 1 ml 0.1 ml — — —<1 × 10⁵ 1 b — 1.1 ml — — — <1 × 10⁵ 1 c 1 ml — YGP 100   3 × 10⁷ <1 ×10⁵ 1 d — 1 ml YGP 100   3 × 10⁷ <1 × 10⁵ 1 e 1 ml — YGP:tet 100 3.75 ×10⁶    1 × 10⁸ 100 f — 1 ml YGP:tet 100 3.75 × 10⁶    1 × 10⁶ — g 1 ml —YGP:tet 100 7.5 × 10⁶  >1 × 10⁸ >10 h — 1 ml YGP:tet 100 7.5 × 10⁶   1 ×10⁷ — i 1 ml — YGP:tet 100 1.5 × 10⁷ >1 × 10⁸ — j — 1 ml YGP:tet 100 1.5× 10⁷ >1 × 10⁸ — k 1 ml — YGP:tet 100   3 × 10⁷ >1 × 10⁸ — l — 1 mlYGP:tet 100   3 × 10⁷ >1 × 10⁸ — m 1 ml — tet − 1.25 100 1.25 μg/ml   1× 10⁶ — n — 1 ml tet − 1.25 100 1.25 μg/ml   1 × 10⁶ 1 o 1 ml — tet −2.5 100 2.5 μg/ml   1 × 10⁷ 3.3 p — 1 ml tet − 2.5 100 2.5 μg/ml 3.3 ×10⁶ — q 1 ml — tet − 5 100 5 μg/ml >1 × 10⁸ — r — 1 ml tet − 5 100 5μg/ml >1 × 10⁸ — s 1 ml — tet − 10 100 10 μg/ml >1 × 10⁸ — t — 1 ml tet− 10 100 10 μg/ml >1 × 10⁸ —

One ml of murine macrophages, J774 (5×10⁵/ml) was combined with YGP,YGP:alginate-tet or tetracycline of various concentration as summarizedin Table 6, above.

The J774 cells were cultured overnight in medium (DMEM containing 10%fetal calf serum without antibiotics or glutamine). The cultures wereincubated with medium alone, tetracycline diluted in medium or particlesdiluted in medium for 1 hour with rotation at 37 degrees Celsius topermit phagocytosis of the particles. The microbial killing assay wasset up in 96 well plates. The cultures were diluted in medium andincubated overnight to allow for metabolism and release of tet fromphagocytosed YGP: alginate-tet particles. Bacterial challenge was addedas indicated in Table 6 and the cultures were incubated 2 hours at 37degrees Celsius in a CO₂ incubator to permit S. aureus phagocytosis andkilling by the J774 murine macrophages. After this incubation, 200 μl LBBroth (Luria-Bertani Broth: 1.0% tryptone, 0.5% yeast extract, 1.0%NaCl) was added to each culture to lyze the macrophages. Cultures wereincubated at 37 degrees Celsius in an incubator to permit outgrowth ofsurviving & aureus. Growth was monitored by change in pH as indicated byphenol red. The effects of YGP, YGP: alginate-tet or tetracycline werecompared. The results are provided in the two right-most columns ofTable 6.

About 7.5×10⁶ YGP: alginate-tet particles produced an effect onmacrophages roughly equivalent to about 2.5 μg/ml tetracycline HCl. Themacrophages alone were relatively less effective than macrophagestreated with tetracycline in either mode, and about as effective asmacrophages treated with empty YGP alone. Macrophages in combinationwith free tetracycline in solution were not much more effective thantetracycline alone. Macrophages treated with YGP:alginate-tet particlesshowed significant synergy. In general, the results demonstrate thatphagosome delivery of tetracycline into J774 macrophage cells enhancesthe killing capacity of J774 macrophage cells for S. aureus.

Example 15 Loading of Protein into YGP

The utility of the delivery system of the present invention for theretention, transport and delivery of therapeutic peptides or proteins,vaccine antigens or other peptides or proteins was evaluated using themixed proteins of fetal calf serum. Yeast cell wall particles used wereYGP, YGP-PEI and YGP-chitosan prepared as described above. Stocksolutions were 45 ng/μl fetal calf serum (FCS) (Fetal Bovine Serum, JRHBiosciences, Lenexa, Kans.), 0.2% PE (Sigma Chemical Co., St. Louis,Mo.) in TE, 0.05 M phosphate buffer, pH 7.2 (P buffer) and 0.05 Mphosphate buffer, pH 7.2, 1 M NaCl (P+salt buffer).

Four μl of FCS were added to 1 mg of YGP, YGP-P or YGP-CN inmicrocentrifuge tubes as indicated in Table 7 and the resulting mixturewas incubated 60 minutes at room temperature to allow the liquid to beabsorbed by the particles. After the incubation, 200 μl phosphate bufferor 200 μl PEI was as indicated in Table 7 and the resulting mixture wasincubated 60 minutes at room temperature. After the incubation, 0.5 mlphosphate buffer was added, and after a further 5 minute incubation, thetubes were sonicated to produce single particles. The particles werepelleted by centrifuging at 10,000 rpm for 10 minutes and thesupernatants were removed to fresh tubes. 0.5 ml 0.05M sodium phosphatebuffer, pH 7.2+1M NaCl was added to the pellets, and after a further 5minute incubation, the tubes were centrifuged at 10,000 rpm for 10minutes and the high salt elution supernatants were removed to freshtubes. The protein content of the supernatants was measured byabsorbance at 280 nm.

TABLE 7 P buffer P + Salt Tube YGP 1° Load 2° Load (μl) buffer (μl) 1 —4 μl FCS 200 μl P buffer 500 500 2 YGP 4 μl FCS 200 μl P buffer 500 5003 YGP 4 μl FCS 200 μl 2% PEI 500 500 4 YGP-PEI 4 μl FCS 200 μl P buffer500 500 5 YGP-CN 4 μl FCS 200 μl P buffer 500 500

The protein loading results are shown in Table 8. YGP particles withouta trapping molecule trapped only 5% of the presented protein. YGPparticles that were loaded first with FCS protein and then exposed toPEI retained 47% of the protein load. YGP particles that were preloadedwith a trapping polymer such as PEI or chitosan before exposure to theprotein load such retained 68% and 60%, respectively, of the proteinload.

TABLE 8 Unbound % Bound % Pay- Trapping Protein Unbound Protein BoundTube YGP load Polymer (ng) Protein (ng) Protein 1 — FCS P buffer 180 100— — 2 YGP FCS P buffer 180 95 10 5 3 YGP FCS 2% PEI 120 63 70 47 4 YGP-FCS P buffer 60 32 130 68 PEI 5 YGP- FCS P buffer 80 40 120 60 CN

The results demonstrate that serum proteins are not effectively loadedand trapped into YGP without trapping polymers. Using YGP that werepreloaded with trapping polymers before exposure to the payload proteinsresulted in increased protein trapping. Alternatively, proteins can betrapped inside YGP by first loading the protein, and then adding asoluble trapping polymer to sequester the protein within the particle.

Example 16 Comparison of Various Methods of Loading DNA into YGP

Several methods of loading salmon sperm DNA into YGP, YGP containing lowmolecular weight (LMW) chitosans or YGP containing high molecular weight(HMW) chitosans were evaluated.

a. Capillary Loading Followed by Ethanol Precipitation

Salmon sperm DNA Sigma, St. Louis, Mo.) was sheared by 40 passes through18 gauge needle and diluted to a concentration of 0.1 mg/ml in 50 mM TE(Tris-HCl, pH 8, 2 mM EDTA). Loading volumes of the DNA solution weredetermined and mixed in centrifuge tubes in duplicate with 100 mgaliquots of YGP, YGP:LMW chitosan or YGP:HMW chitosan as in Example 2and incubated 1 hour. The incubated mixtures were ethanol precipitatedby adding 1.5 ml ethanol to each tube. The insoluble products werecollected by centrifugation at 2,000 rpm for 10 minutes. 10 ml TE wasadded to each tube, incubated for 1 hr at 37 degrees Celsius,centrifuged 2,000 rpm for 10 minutes to sediment the insoluble YGP andthe DNA content of the supernatant was determined by absorbance at 260nm. The amount of DNA remaining in the YGP was calculated.

b. DNA Loading by Absorption

Loading volumes of the DNA solution were mixed in centrifuge tubes induplicate with 100 mg aliquots of YGP, YGP:LMW chitosan or YGP:HMWchitosan as in Example 4a and incubated 1 hour. 10 ml TE was added toeach tube, incubated for 1 hr at 37 degrees Celsius, centrifuged 2,000rpm for 10 minutes to sediment the insoluble YGP. The DNA content of thesupernatant was determined by absorbance at 260 nm. The amount of DNAremaining in the YGP was calculated.

c. DNA Loading by CTAB Trapping

Loading volumes of the DNA solution were mixed in centrifuge tubes induplicate with 100 mg aliquots of YGP, YGP:LMW chitosan or YGP:HMWchitosan as in Example 4 and incubated 1 hour. The incubated mixtureswere precipitated by adding 1.5 ml 2% hexadecyltrimethylammoniumbromide(also known as cetyltrimethylammonium bromide or CTAB) solution to eachtube. 10 ml TE was added to each tube, which was incubated for 1 hr at37 degrees Celsius, and centrifuged 2,000 rpm for 10 minutes to sedimentthe insoluble YGP. The DNA content of the supernatant was determined byabsorbance at 260 nm. The amount of DNA remaining in the YGP wascalculated.

The amount of DNA remaining in the YGP was calculated.

The results are presented in Table 9, below.

TABLE 9 % DNA bound in YGP YGP:LMW YGP:HMW Method YGP chitosan chitosanDirect Loading  <1% 32% 70% Direct Loading +  <1% Not done Not doneEthanol Direct Loading >99% >99% 99% CTAB trapping Absorption Loading <1%  5% 12%

Simple DNA loading or precipitation failed to effectively load and trapDNA into the YGP. In contrast, the use of the cationic trapping polymer,chitosan, resulted in the formation of chitosan-DNA complexes that cantrap DNA inside YGP. In addition, the cationic agent CTAB can beeffectively used to trap loaded DNA into YGP.

Example 17 DNA Loading and Trapping

Fluorescent salmon sperm DNA was prepared by mixing 1 ml of a 1 mg/mlsolution of salmon sperm DNA in 0.1M carbonate buffer pH 9.2 with 100 μlof a 1 mg/ml suspension of DTAF in 10 mM carbonate buffer ph 9.2. Afterovernight incubation at 37 degrees Celsius, 200 μl 1M Tris-HCl pH 8.3was added and incubated for 15 minutes at room temperature. Then, 100 μl1M NaCl and 3 mls ethanol were added to ethanol precipitate the DNA.After storage at −20 C overnight, the ethanol precipitate was collectedby centrifugation at 10,000 rpm 15 minutes. The ethanol precipitate waswashed with 70% ethanol until supernatant was clear and resuspended in 1ml TE.

The YGP suspensions were incubated for 30 minutes at room temperature.After the incubation, 0.45 ml 95% ethanol was added to one set (YGP,YGP-P, YGP-Chitosan) of three tubes, 0.2 ml 2% PEI was added to two setsof three tubes and 0.2 ml 2% CTAB was added to another set of threetubes. After 30 minutes incubation at room temperature, 0.2 ml 2% CTABwas added to one set of the PEI tubes and incubation proceeded for afurther 30 minutes. Ethanol (1 ml, 95%) was added and the YGPs werestored overnight at −20 degrees Celsius. The YGP suspensions were washedwith 70% ethanol and resuspended in 0.5 ml PBS. Results were evaluatedby fluorescence microscopy, and are shown in Table 10.

TABLE 10 Fluorescence Microscopy Particle Treatment YGP pelletObservation YGP ethanol White Not fluorescent YGP-CN ethanol YellowInternal particle fluorescence YGP-P ethanol Yellow Internal particlefluorescence YGP 2% PEI Yellow Internal particle fluorescence YGP-CN 2%PEI Yellow Weak internal particle fluorescence YGP-P 2% PEI Yellow Weakinternal particle fluorescence YGP 2% CTAB Yellow Internal particlefluorescence YGP-CN 2% CTAB Yellow Strong internal particle fluorescenceYGP-P 2% CTAB Yellow Strong internal particle fluorescence YGP 2% PEI/2%CTAB Yellow Strong internal particle fluorescence YGP-CN 2% PEI/2% CTABYellow Internal particle fluorescence YGP-P 2% PEI/2% CTAB YellowInternal particle fluorescence

No significant trapping of fluorescent-labeled DNA occurred if onlysimple ethanol precipitation without a trapping polymer was used,demonstrating that the prior art technology is not effective as a DNAdelivery system. Fluorescent-labeled DNA was clearly being trapped bycationic trapping polymers PEI or chitosan, or with the cationicdetergent CTAP inside YGP particles. The best DNA trapping occurred whena combination of trapping polymer and CTAB was used, such asYGP:PEI:DNA:CTAB, YGP:chitosan:DNA:CTAB or YGP:DNA:PEI:CTAB.

Example 18 Fluorescently Labeled Plasmid DNA Loading and Trapping

YGP containing pIRES plasmid was prepared for transfection andexpression of encoded EGFP in J774 cells, a murine macrophage derivedcell line. Cationic trapping agents used included cationic polymers suchas polyethylenimine (PEI), CytoPure™, a proprietary, commerciallyavailable, water-soluble cationic polymer transfection reagent(Qbiogene, Inc., CA), chitosan and a cationic detergenthexadecyltrimethyl-ammoniumbromide (CTAB). A preferred PEI is JetPEI, acommercially available linear polyethylenimine cationic polymertransfection reagent (Qbiogene, Inc., CA).

pIRES-EGFP (Clonetech, Calif.) contains the internal ribosome entry site(IRES) of the encephalomyocarditis virus (ECMV) between the MCS and theEGFP (enhanced green fluorescent protein) coding region. This permitsboth the gene of interest (cloned into the MCS) and the EGFP gene to betranslated from a single bicistronic mRNA. pIRES-EGFP is designed forthe efficient selection (by flow cytometry or other methods) oftransiently transfected mammalian cells expressing EGFP and anotherprotein of interest. To optimize the selection of cells expressing highlevels of the protein of interest, pIRES-EGFP utilizes a partiallydisabled IRES sequence (1). This attenuated IRES leads to a reduced rateof translation initiation at the EGFP start codon relative to that ofthe cloned gene. This enables the selection of those cells in which themRNA, and hence the target protein, is produced at high levels tocompensate for a suboptimal rate of translation of EGFP. This vector canalso be used to express EGFP alone or to obtain stably transfected celllines without time-consuming drug and clonal selection. EGFP is ared-shifted variant of wild-type GFP that has been optimized forbrighter fluorescence and higher expression in mammalian cells.(Excitation maximum=488 nm; emission maximum=509 nm) EGFP encodes theGFPmut1 variant, which contains the amino acid substitutions Phe-64 toLeu and Ser-65 to Thr. These mutations increase the brightness andsolubility of GFP, primarily due to improved protein folding propertiesand efficiency of chromophore formation. EGFP also contains an openreading frame composed almost entirely of preferred human codons. Thisleads to more efficient translation and, hence, higher expression levelsin eukaryotic cells, relative to wild type GFP.

Solutions prepared were: pIRES EGFP plasmid DNA, 0.72 μg/μl in water,0.2% w/v PEI (Sigma) in TE, 2 μl CytoPure (Qbiogene)+48 μl 0.15M NaCl, 2μl JetPEI (Qbiogene)+48 μl TE, 0.2% Spermidine in TE, 2% (aq) CTAB andphosphate buffered saline (PBS).

Fluorescent pIRES plasmid DNA was prepared by mixing 1 ml of a 1 mg/mlsolution of pIRES DNA in 0.1M carbonate buffer pH 9.2 with 100 μl of a 1mg/ml suspension of DTAF in 10 mM carbonate buffer pH 9.2. Afterovernight incubation at 37 degrees Celsius, 200 μl 1M Tris-HCl pH 8.3was added and incubated for 15 minutes at room temperature. Then 100 μl1M NaCl and 3 ml ethanol were added to ethanol precipitate the DNA.After storage at −20 degrees Celsius overnight, the ethanol precipitatewas collected by centrifugation at 10,000 rpm 15 minutes. The ethanolprecipitate was washed with 70% ethanol until supernatant was clear andresuspended in 1 ml TE.

The YGP suspensions were incubated for 30 minutes at room temperature.After the incubation, 0.45 ml 95% ethanol was added to one set (YGP,YGP-P, YGP-Chitosan) of three tubes, 0.2 ml 2% PEI was added to two setsof three tubes and 0.2 ml 2% CTAB was added to another set of threetubes. After 30 minutes incubation at room temperature, 0.2 ml 2% CTABwas added to one set of the PEI tubes and incubation proceeded for afurther 30 minutes. Ethanol (1 ml, 95%) was added and the YGPs werestored overnight at −20 degrees Celsius. The YGP suspensions were washedwith 70% ethanol and resuspended in 0.5 mil PBS.

J774 murine macrophages were plated in six well plates at a density of2.5×10⁵ cells per well and incubated overnight as described in Example14. The transfections were performed as summarized in Table 11. Theparticles were added to the culture medium at a 10 particle per cellratio and the plates were swirled to distribute particles. The cellswere incubated for 4 hours. At end of the incubation period, the culturemedium was removed, the cells were washed with PBS and fixed in 0.4%formalin in PBS.

TABLE 11 0.2% 0.2% Chitosan pIRES vol YGP PEI in Acetate 2% Tube μg/μlμl mg in TE buffer pH 5.5 CTAB Ethanol 1 — — 1 200 μl — 200 μl 800 μl 2— — 1 — 200 μl 200 μl 800 μl 3 1.8 4 1 200 μl — 200 μl 800 μl 4 1.8 4 1— 200 μl 200 μl 800 μl

Fluorescent DNA-containing particles and J774 cells incubated withfluorescent DNA-containing particles were evaluated by fluorescencemicroscopy, and results are summarized in Table 12 and shown in FIGS. 3Aand 3B.

TABLE 12 Particle Color of Microscopic Examintation Type TreatmentPellet of Particles YGP ethanol White Not fluorescent YGP-CN ethanolYellow Intracellular fluorescent particles YGP-P ethanol YellowIntracellular fluorescent particles YGP 2% PEI Yellow Intracellularfluorescent particles YGP-CN 2% PEI Yellow Intracellular fluorescentparticles YGP-P 2% PEI Yellow Intracellular fluorescent particles YGP 2%CTAB Yellow Intracellular fluorescent particles YGP-CN 2% CTAB YellowIntracellular fluorescent particles YGP-P 2% CTAB Yellow Intracellularfluorescent particles YGP 2% PEI/2% CTAB Yellow Figures 3A & 3B;strongly fluorescent Intracellular particles YGP-CN 2% PEI/2% CTABYellow Intracellular fluorescent particles YGP-P 2% PEI/2% CTAB YellowIntracellular fluorescent particles

FIG. 3A is a reversed contrast (negative) grayscale image of a colorlight photomicrograph of cells exposed to YGP particles loaded withfluorescent labeled pIRES plasmid with PEI as the cationic trappingpolymer and CTAB as a cationic detergent, indicating a cell 310. FIG. 3Bis a reversed contrast (negative) grayscale image of a colorfluorescence photomicrograph of the same field of cells showing brightstaining representing fluorescent YGP particles containing fluorescentplasmid DNA internalized by the same cell 310 indicated in FIG. 3B.

Example 19 EGFP Expression by J774 Murine Macrophages Incubated withYGP:pIRES

The pIRES plasmid DNA was not fluorescently labeled in this Example,rather the functional expression of the green fluorescent protein (GFP)encoded by pIRES was used as a demonstration of uptake of loaded yeastcell wall particles, intracellular release of the pIRES DNA andexpression of the GFP as evidenced by the production of fluorescence.

The YGP: pIRES formulations were prepared as summarized in Table 12,below. DNA was prepared from dilutions in deionized water of 1 mg/mlstock. The indicated amount of DNA solution was added to YGP andincubated for at least 30 minutes to allow for liquid absorption. Theindicated amount of 0.2% PEI in TE or 0.2% chitosan in acetate bufferwas added and the mixture was allowed to incubate for 5 minutes beforesonication to produce single particles. After a further incubation of atleast 30 minutes; the indicated amount of 2% CTAB was added. After anadditional 5 minute incubation, the tubes were vortex mixed andincubated again for at least 30 minutes. The indicated amount of 95%ethanol was added. Each tube was then mixed and stored at −20 Celsiusovernight. The YGP:pIRES formulated particles were then centrifuged,washed twice in 70% ethanol, collected by centrifugation at 10,000 rpmfor 5 minutes, resuspended in 0.5 ml sterile PBS and sonicated toproduce single particles. The number of particles per ml was counted andeach formulation was and stored at −20 degrees Celsius.

J774 murine macrophages were plated in 6 well plates at a density of2.5×10⁵ cells per well and incubated overnight as described in Example14. The transfections were performed as summarized in Table 11, above.The particles were added to the culture medium at a 10 particle per cellratio and the plates were swirled to distribute particles. The cellswere fed daily and incubated for 2 days. At end of the incubationperiod, the culture medium was removed the cells were washed with PBSand fixed in 0.4% formalin in PBS.

The results are summarized in Table 13 and shown in FIGS. 4A-C. Cellswere examined using fluorescence microscopy. Eighty nine percent of J774cells took up YGP-F particles (Table 13, well IB, FIG. 4A). EGFPexpression was evident in >80% of J774 cells as punctate fluorescence invacuoles in wells 1E (FIG. 4B) and 1F (FIG. 4C).

TABLE 13 Well Description YGP/Cell volume Appearance 1A No Treatment 0 —No detectible GFP Control fluorescent particles 1B YGPF Particle 10  10μl 1/10 Figure 4A, showing Uptake Control phagocytosis of fluorescentYGFP particles 1C YGP empty 10  11 μl 1/10 No detectible GFP PEI/CTABControl fluorescent particles 1D YGP empty 10   5 μl 1/10 No detectibleGFP Chitosan/CTAB fluorescent particles Control 1E YGP plRES 10  10 μl1/10 Figure 4B, showing PEI/CTAB fluorescent GFP expression in cells 1FYGP plRES 10 6.5 μl 1/10 Figure 4C, showing Chitosan/CTAB fluorescentGFP expression in cells

FIG. 4A is a reversed contrast (negative) grayscale image of a colorfluorescence photomicrograph of cells, e.g., an indicated cell 410,exposed to fluorescent labeled YGP particles, FIG. 4B is a reversedcontrast (negative) grayscale image of a color fluorescencephotomicrograph of cells, e.g., an indicated cell 420, expressing GFPfrom pIRES DNA delivered by YGP with a cationic trapping polymerpolyethylenimine (PEI) and cationic detergenthexadecyltrimethylammoniumbromide (also known as cetyltrimethylammoniumbromide or CTAB) and FIG. 4C is a reversed contrast (negative) grayscaleimage of a color fluorescence photomicrograph of cells, e.g., anindicated cell 430, expressing GFP from pIRES DNA delivered by YGP witha cationic trapping polymer chitosan and cationic detergent CTAB.

Example 20 Fluorescent DNA, Oligonucleotide and siRNA OligonucleotideDelivery into J774 Cells Using YGP-Cation Trapping Polymer Technology

The following materials were used: YGP:Fluorescent salmon spermDNA:PEI:CTAB particles, YGP:Fluorescent oligonucleotide:PEI:CTABparticles, and YGP:Fluorescent siRNA:PEI:CTAB. The fluorescentoligonucleotide was an 18 mer synthesized by Sigma Genosys with afluorescein residue attached to the 5′ end:

SEQ ID NO: 1 5′ Fluorescein-TTGGTCATCCATGGCTCT 3′.The fluorescent siRNA was a 21 mer non-silencing control siRNAsynthesized with a fluorescein residue attached to the 5′ end (Qiagen,Valencia, Calif., Catalog No. 1022079):

SEQ ID NO: 2 5′ Fluorescein-UUCUCCGAACGUGUCACGUdTdT 3′.

J774 murine macrophages were plated in 6 well plates at a density of2.5×10⁵ cells per well and incubated overnight as described in Example14. The transfections were performed as summarized in Table 14. Thecontrol and polynucleotide-loaded particles were added to the culturemedium and the plates were swirled to distribute particles. The cellswere fed daily and incubated for 24 hours. At end of the incubationperiod, the culture medium was removed the cells were washed with PBSand fixed in 0.4% formalin in PBS.

TABLE 14 YGP/Cell Well Cells Ratio Particles 1A J774 0 — 1B J774 10 YGPF1C J774 10 YGP DNAF 1D J774 10 YGP oligoF 1E J774 10 YGP RNAiF

The results are illustrated in FIGS. 5A-I. Cells were examined usingfluorescence microscopy and FACS. 92% of J774 cells took up YGP-Fparticles (Table 14, well IB, FIG. 5A). Fluorescent oligonucleotide (SEQID NO:1) delivery was evident in >80/a of J774 cells as punctateendosomal fluorescence and diffuse cytoplasmic fluorescence. Fluorescentnon-silencing siRNA (SEQ ID NO:1) delivery was evident in >80% of J774cells as punctate endosomal fluorescence and diffuse cytoplasmicfluorescence.

FIG. 5A is a reversed contrast (negative) grayscale image of a colorcombined light and fluorescence photomicrograph of cells, e.g., anindicated cell 510, exposed to fluorescent labeled YGP particles; FIG.5B is a graphic representation of the results of a fluorescenceactivated cell sorting (FACS) study showing a major peak 520representing the distribution of signals from cells that haveinternalized fluorescent labeled YGP particles and a minor peak 530representing the distribution of signals from cells without fluorescentlabeled YGP particles; FIG. 5C is a reversed contrast (negative)grayscale image of a color light photomicrograph of cells, e.g., anindicated cell 540, exposed to YGP particles containing fluorescentlabeled DNA, a cationic trapping polymer PEI and cationic detergentCTAB; FIG. 5D is a reversed contrast (negative) grayscale image of acolor fluorescence photomicrograph of the same field of cells showingthe same indicated cell 540, FIG. 5E is a graphic representation of theresults of a FACS study showing a major peak 610 representing thedistribution of signals from cells that have internalized YGP particleswith fluorescent DNA payload and a shoulder 620 representing thedistribution of signals from cells without YGP particles; FIG. 5F is areversed contrast (negative) grayscale image of a color lightphotomicrograph of cells, e.g., an indicated cell 710, incubated withYGP particles containing fluorescent labeled antisense RNA, PEI andCTAB; FIG. 5G is a reversed contrast (negative) grayscale image of acolor fluorescence photomicrograph of the same field of cells showingthe same indicated cell 710 containing internalized YGP particles withfluorescent antisense RNA payload; FIG. 5H is a reversed contrast(negative) grayscale image of a color light micrograph of cells, e.g.,an indicated cell 810, incubated with YGP particles containingfluorescent labeled siRNA, PEI and CTAB and FIG. 5I is a reversedcontrast (negative) grayscale image of a color fluorescencephotomicrograph of the same field of cells showing the same indicatedcell 810 containing internalized YGP particles with fluorescent RNAipayload.

In summary, fluorescent DNA, oligonucleotide or siRNA payloads loadedinto YGP using a cationic trapping polymer efficiently delivers thepayload into J774 cells. Payloads are released from the endosomalcompartment within 24 hours into the cytoplasm and nuclear compartments.

The claims should not be read as limited to the described order orelements unless stated to that effect. Therefore, all embodiments thatcome within the scope and spirit of the following claims and equivalentsthereto are claimed as the invention.

1. A particulate delivery system comprising an extracted yeast cell wallcomprising beta-glucan and a payload trapping molecule.
 2. Theparticulate delivery system of claim 1 further comprising a payloadmolecule, wherein the payload molecule and the payload trapping moleculeare soluble in the same solvent system.
 3. The particulate deliverysystem of claim 2 wherein the solvent system comprises water.
 4. Theparticulate delivery system of claim 1 wherein the extracted yeast cellwall comprises one or more of the following: (a) less than 90 weightpercent beta-glucan; (b) more than 50 weight percent chitin; (c) morethan 30 weight percent mannan; and (d) more than 1 weight percentprotein. 5.-7. (canceled)
 8. The particulate delivery system of claim 1wherein the payload trapping molecule is; (a) a polysaccharide selectedfrom the group consisting of agarose, an alginate, a xanthan, a dextran,a chitosan, a galactomannan gum, a derivative thereof and a mixturethereof; (b) a polyacrylamide: (c) a polyamide; (d) selected from thegroup consisting of a cationic polymer, an anionic polymer, a cationicdetergent, an anionic detergent and a mixture thereof; (e) a cationicpolymer selected from the group consisting of chitosan, polyethyleniminepoly-L-lysine, a protein, a polypeptide, a short synthetic peptide, ahelical amphiphilic peptide, a cationic dendrimers, glucaramide polymer,a N-substituted glycine oligomer, poly(2-methyl-acrylic acid2-[(2-dimethylamino)-ethyl)-methyl-amino]-ethyl ester),poly(2-dimethylamino ethyl)-methacrylate and mixtures thereof. (f)hexadecyltrimethylammoniumbromide; (g) selected from the groupconsisting of a cationic polyelectrolyte, an anionic polyelectrolyte andan amphoteric polyelectrolyte; (h) a cationic polyelectrolyte selectedfrom the group consisting of a copolymer of vinyl pyrollidone andquaternary methyl methacrylate, a substituted polyacrylamide,polyethyleneimine, polypropyleneimine, a polyamine homopolymer, apolyamine co-polymer, polydiallyl dimethyl ammonium chloride,substituted dextrans; modified guar gum, a substituted protein, apolyamino acid, spermine and spermidine; or (i) an anionicpolyelectrolyte selected from the group consisting of a copolymer ofmethyl vinyl ether and maleic anhydride, a copolymer of methyl vinylether and maleic acid, alginic acid a carboxymethyl cellulose, asubstituted polyacrylamide, a polyacrylic acid, a polystyrene sulfonicacid, a dextran sulphates, a substituted saccharide, heparin andpharmaceutically acceptable salts. 9.-19. (canceled)
 20. The particulatedelivery system of claim 2 wherein the payload molecule is selected fromthe group consisting of a polynucleotide, a peptide, a protein, a smallorganic active agent, a small inorganic active agent and a mixturethereof.
 21. The particulate delivery system of claim 20 wherein thepolynucleotide is selected from the group consisting of anoligonucleotide, an antisense construct, a siRNA, an enzymatic RNA, arecombinant DNA construct and a mixture thereof.
 22. The particulatedelivery system of claim 21 wherein the recombinant DNA construct is anexpression vector comprising a control element operatively linked to anopen reading frame encoding a protein.
 23. The particulate deliverysystem of claim 22 wherein the protein encoded by the open reading frameis a structural protein, a protein having enzymatic activity, a membraneprotein, a DNA binding protein or a signaling protein.
 24. (canceled)25. The particulate delivery system of claim 19 wherein thepolynucleotide comprises a nucleotide sequence that restores thefunction of an absent, defective or inhibited gene.
 26. The particulatedelivery system of claim 22 wherein the protein encoded by the openreading frame is a protein that produces a therapeutic effect in anindividual having a genetic disorder.
 27. The particulate deliverysystem of claim 25 wherein the genetic disorder is Aarskog-Scottsyndrome, Aase syndrome, achondroplasia, acrodysostosis, addiction,adreno-leukodystrophy, albinism, ablepharon-macrostomia syndrome,alagille syndrome, alkaptonuria, alpha-1 antitrypsin deficiency,Alport's syndrome, Alzheimer disease, asthma, autoimmune polyglandularsyndrome, androgen insensitivity syndrome, Angelman syndrome, ataxia,ataxia telangiectasia, atherosclerosis, attention deficit hyperactivitydisorder (ADHD), autism, baldness, Batten disease, Beckwith-Wiedemannsyndrome, Best disease, bipolar disorder, brachydactyl), breast cancer,Burkitt lymphoma, chronic myeloid leukemia, Charcot-Marie-Tooth disease,Crohn's disease, cleft lip, Cockayne syndrome, Coffin Lowry syndrome,colon cancer, congenital adrenal hyperplasia, Cornelia de Langesyndrome, Costello syndrome, Cowden syndrome, craniofrontonasaldysplasia, Crigler-Najjar syndrome, Creutzfeldt-Jakob disease, cysticfibrosis, deafness, depression, diabetes, diastrophic dysplasia,DiGeorge syndrome, Down's syndrome, dyslexia, Duchenne musculardystrophy, Dubowitz syndrome, ectodermal dysplasia, Ellis-van Creveldsyndrome, Ehlers-Danlos, epidermolysis bullosa, epilepsy, essentialtremor, familial hypercholesterolemia, familial Mediterranean fever,fragile X syndrome, Friedreich's ataxia, Gaucher disease, glaucoma,glucose galactose malabsorption, glutaricaciduria, gyrate atrophy,Goldberg Shprintzen syndrome (velocardiofacial syndrome), Gorlinsyndrome, Hailey-Hailey disease, hemihypertrophy, hemochromatosis,hemophilia, hereditary motor and sensory neuropathy (HMSN), hereditarynon polyposis colorectal cancer (HNPCC), Huntington's disease,immunodeficiency with hyper-IgM, juvenile onset diabetes, Klinefelter'ssyndrome, Kabuki syndrome, Leigh's disease, long QT syndrome, lungcancer, malignant melanoma, manic depression, Marfan syndrome, Menkessyndrome, miscarriage, mucopolysaccharide disease, multiple endocrineneoplasia, multiple sclerosis, muscular dystrophy, myotrophic lateralsclerosis, myotonic dystrophy, neurofibromatosis, Niemann-Pick disease,Noonan syndrome, obesity, ovarian cancer, p53 tumor suppressor,pancreatic cancer, Parkinson disease, paroxysmal nocturnalhemoglobinuria, Pendred syndrome, peroneal muscular atrophy,phenylketonuria (PKU), polycystic kidney disease, Prader-Willi syndrome,primary biliary cirrhosis, prostate cancer, REAR syndrome, Refsumdisease, retinitis pigmentosa, retinoblastoma, Rett syndrome, Sanfilipposyndrome, schizophrenia, severe combined immunodeficiency, sickle cellanemia, spina bifida, spinal muscular atrophy, spinocerebellar atrophy,SRY: sex determination, sudden adult death syndrome, Tangier disease,Tay-Sachs disease, thrombocytopenia absent radius syndrome,Townes-Brocks syndrome, tuberous sclerosis, Turner syndrome, Ushersyndrome, von Hippel-Lindau syndrome, Waardenburg syndrome, Weaversyndrome, Werner syndrome, Williams syndrome, Wilson's disease,xeroderma pigmentosum or Zellweger syndrome.
 28. The particulatedelivery system of claim 20 wherein the protein is selected from thegroup consisting of growth hormone, prolactin, placental lactogen,erythropoietin, thrombopoietin, interleukin-2, interleukin-3,interleukin-4, interleukin-5, interleukin-6, interleukin-7,interleukin-9, interleukin-10, interleukin-11, interleukin-12 (p35subunit), interleukin-13, interleukin-15, oncostatin M, ciliaryneurotrophic factor, leukemia inhibitory factor, alpha interferon, betainterferon, gamma interferon, omega interferon, tau interferon,granulocyte-colony stimulating factor, granulocyte-macrophage colonystimulating factor, macrophage colony stimulating factor,cardiotrophin-1growth hormone releasing factor; parathyroid hormone;thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin; insulinA-chain; insulin B-chain; proinsulin; follicle stimulating hormone;calcitonin; luteinizing hormone; glucagon; factor VIIIC, factor IXtissue factor, von Willebrands factor, Protein C, atrial natriureticfactor, lung surfactant, urokinase, tissue-type plasminogen activator,bombazine, thrombin, alpha tumor necrosis factor, beta tumor necrosisfactor, enkephalinase; RANTES, human macrophage inflammatory protein,serum albumin, mullerian-inhibiting substance, relaxin A-chain, relaxinB-chain, prorelaxin, mouse gonadotropin-associated peptide, DNase,inhibin, activin, vascular endothelial growth factor, a hormonereceptor, a growth factors receptor, an integrin, protein A, protein D,a rheumatoid factor, a neurotrophic factor, bone-derived neurotrophicfactor (BDNF), neurotrophin-3, neurotrophin-4, neurotrophin-5, orneurotrophin-6, NGF-beta, platelet-derived growth factor (PDGF); alphafibroblast growth factor, beta alpha fibroblast growth factor, epidermalgrowth factor, transforming growth factor-alpha, transforming growthfactor-beta1, transforming growth factor-beta2, transforming growthfactor-beta3, transforming growth factor-beta4, transforming growthfactor-beta5, insulin-like growth factor-I, insulin-like growthfactor-II, des(1-3)-insulin-like growth factor-I, a insulin-like growthfactor binding protein, CD3, CD4, CD8, CD19, CD20, an osteoinductivefactor, an immunotoxin, a bone morphogenetic protein, a T-cell receptor,surface membrane proteins, decay accelerating factor, a viral antigen, atransport protein, homing receptor, an addressing, a regulatory protein,an immunoadhesin, an antibody and biologically active fragments orvariants thereof.
 29. The particulate delivery system of claim 22wherein the protein encoded by the open reading frame is selected fromthe group consisting of growth hormone, prolactin, placental lactogen,erythropoietin, thrombopoietin, interleukin-2, interleukin-3,interleukin-4, interleukin-5, interleukin-6, interleukin-7,interleukin-9, interleukin-10, interleukin-11, interleukin-12 (p35subunit), interleukin-13, interleukin-15, oncostatin M, ciliaryneurotrophic factor, leukemia inhibitory factor, alpha interferon, betainterferon, gamma interferon, omega interferon, tau interferon,granulocyte-colony stimulating factor, granulocyte-macrophage colonystimulating factor, macrophage colony stimulating factor,cardiotrophin-1 growth hormone releasing factor; parathyroid hormone;thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin; insulinA-chain; insulin B-chain; proinsulin; follicle stimulating hormone;calcitonin; luteinizing hormone; glucagon; factor VIIIC, factor IXtissue factor, von Willebrands factor, Protein C, atrial natriureticfactor, lung surfactant, urokinase, tissue-type plasminogen activator,bombazine, thrombin, alpha tumor necrosis factor, beta tumor necrosisfactor, enkephalinase; RANTES, human macrophage inflammatory protein,serum albumin, mullerian-inhibiting substance, relaxin A-chain, relaxinB-chain, prorelaxin, mouse gonadotropin-associated peptide, DNase,inhibin, activin, vascular endothelial growth factor, a hormonereceptor, a growth factors receptor, an integrin, protein A, protein D,a rheumatoid factor, a neurotrophic factor, bone-derived neurotrophicfactor (BDNF), neurotrophin-3, neurotrophin-4, neurotrophin-5, orneurotrophin-6, NGF-beta, platelet-derived growth factor (PDGF); alphafibroblast growth factor, beta alpha fibroblast growth factor, epidermalgrowth factor, transforming growth factor-alpha, transforming growthfactor-beta1, transforming growth factor-beta2, transforming growthfactor-beta3, transforming growth factor-beta4, transforming growthfactor-beta5, insulin-like growth factor-I, insulin-like growthfactor-II, des(1-3)-insulin-like growth factor-I, a insulin-like growthfactor binding protein, CD3, CD4, CD8, CD19, CD20, an osteoinductivefactor, an immunotoxin, a bone morphogenetic protein, a T-cell receptor,surface membrane proteins, decay accelerating factor, a viral antigen, atransport protein, homing receptor, an addressin, a regulatory protein,an immunoadhesin, an antibody and biologically active fragments orvariants thereof.
 30. The particulate delivery system of claim 20wherein the small organic active agent is: (a) an oligomer ofheterocyclic polyamides that binds to the minor groove of doublestranded DNA in a sequence specific manner; (b) an oligomer havingmonomeric subunits selected from the group consisting ofN-methylimidazole carboxamide, N-methylpyrrole carboxamide, beta-alanineand dimethylaminopripylamide; (c) a contraceptive agent, agastrointestinal therapeutic agent, a non-steroidal antifertility agent,a parasympathomimetic agent, a psychotherapeutic agent, a majortranquilizer, a minor tranquilizer, a rhinological decongestant, asedative-hypnotic, a steroid, a sulfonamide, a vaccine; a vitamin, anutrient, an antimalarial, an anti-migraine agent, an anti-Parkinsonagent, an anti-spasmodic, an anticholinergic agent, an antitussive, abronchodilator, a cardiovascular agent; an anti-hypertensive agent, acoronary vasodilator, an organic nitrate, an alkaloid, an analgesic, anarcotic, an anti-cancer agent, an anti-convulsant, an anti-emetic, ananti-inflammatory agent, a cytotoxic drug or an antibiotic; or (d) anantibiotic selected from the group consisting of a cephalosporin,chloramphenical, gentamicin, kanamycin A, kanamycin B, a penicillin,ampicillin, streptomycin A, antimycin A, chloropamtheniol,metronidazole, oxytetracycline, penicillin G, a tetracycline andmixtures thereof. 31-33. (canceled)
 34. An article of manufacturecomprising a first container containing a payload molecule selected fromthe group consisting of a nucleic acid composition, protein composition,small organic molecule and mixtures thereof, a second containercontaining the particulate delivery system of claim 1, and instructionsfor use.
 35. A pharmaceutical composition comprising the particulatedelivery system of claim 1, comprising a payload molecule selected fromthe group consisting of a polynucleotide, a protein, a small organicmolecule and a mixture thereof, and a pharmaceutically acceptableexcipient.
 36. A method of delivering a payload molecule to a cellcomprising the steps of: providing an extracted yeast cell wallcomprising beta-glucan, the yeast cell wall defining an internal space;contacting the extracted yeast cell wall with a payload molecule whereinthe payload molecule becomes at least partially enclosed within theinternal space; contacting the extracted yeast cell wall with a payloadtrapping molecule wherein the payload trapping molecule at leastpartially confines the payload molecule within the extracted yeast cellwall to form a particulate delivery system, wherein the payload moleculeand the payload trapping molecule are soluble in the same solventsystem; and contacting a cell with the particulate delivery system.37.-74. (canceled)
 75. A method of making a particulate delivery systemcomprising the steps of: providing an extracted yeast cell wallcomprising beta-glucan, the yeast cell wall defining an internal space;contacting the extracted yeast cell wall with a payload molecule whereinthe payload molecule becomes associated with extracted yeast cell wall;and contacting the extracted yeast cell wall with a payload trappingmolecule wherein the payload trapping molecule stabilizes theassociation of the payload molecule and the extracted yeast cell wall toform a particulate delivery system. 76.-89. (canceled)
 90. A method ofexposing an individual to an antigen comprising the step of contacting aphagocytic cell of the individual with the particulate delivery systemof claim 1 comprising a payload molecule, wherein the payload moleculeis an antigenic molecule. 91.-104. (canceled)
 105. A method ofimmunizing an individual against a hyperproliferative disease comprisingthe step of contacting cells of said individual with the particulatedelivery system of claim 1 comprising a payload molecule that is apolynucleotide comprising a control element operatively linked to anopen reading frame encoding a peptide that comprises an epitopeidentical to, or substantially similar to, an epitope displayed on ahyperproliferative disease-associated protein, wherein encoded peptideis capable of being expressed in the cells of the individual.
 106. Amethod of treating an individual suffering from a genetic diseasecomprising the step of contacting cells of said individual with theparticulate delivery system of claim 1 comprising a payload moleculethat is a polynucleotide comprising a nucleotide sequence that restoresthe activity of an absent, defective or inhibited gene.
 107. (canceled)